Enzyme-containing granules and process for the production thereof

ABSTRACT

The present invention relates to enzyme-containing granules comprising (a) an enzyme and (b) a core which intrinsically is capable of absorbing at least 5% w/w (based on the weight of the core) of water and to processes for the production of such granules comprises (a) contacting absorbent cores, capable of absorbing at least 5% w/w (based on the weight of the core) of water, with a liquid medium, such as an aqueous medium, containing an enzyme in dissolved and/or dispersed form, the amount of the liquid medium employed being such that substantially no attendant agglomeration of the resulting product occurs; and (b) at least partially removing volatile components of the liquid medium from the resulting product.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/DK97/00161 filed Apr. 4, 1997and claims priority under 35 U.S.C. 119 of Danish applications Ser. No.0420/96 and 0759/96 filed Apr. 12, 1996 and Jul. 5, 1996, respectively,and of U.S. application Ser. No. 60/029,738 filed Oct. 23, 1996, thecontents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to an enzyme-containing granulate (made upof enzyme-containing granules or particles) with extremely low tendencyto dust formation, to a process for producing such a granulate, and tothe use of said granulate for a number of industrial applications.

BACKGROUND OF THE INVENTION

The industrial use of enzymes, notably enzymes of microbial origin, hasbecome increasingly common. Enzymes are used in numerous industries,including, for example, the starch-processing industry and the detergentindustry. It is well known that the use of enzymes, particularlyproteolytic enzymes, in the detergent industry has given rise toindustrial hygiene concerns for detergent factory workers, particularlydue to the health risks (including the risk of allergy development)associated with any formation of enzyme-containing dust which may occur.

Since the introduction of enzymes into the detergent industry, a lot ofeffort has been devoted to improving the granulation and coating ofenzymes so as to reduce enzyme dust formation.

One type of process for producing an enzyme-containing granule comprisescoating the surface of a core with an enzyme followed by an outer layercoating. U.S. Pat. No. 5,324,649 describes coating of the surface of anonpareil core with an enzyme followed by an outer layer coating. U.S.Pat. No. 4,689,297 and EP 0 532 777 describe a process which comprisesapplying an enzyme on the surface of a salt crystal based core or anon-pareil core, by spraying the enzyme onto the core in a fluid-bedfollowed by an outer layer coating.

Yet another type of process essentially comprises: (i) mixing an enzymewith suitable granulation components (preferably as dry matter), such asfiller, binder, fibrous material and a granulation agent (e.g. water) ina granulator (e.g. a mixer), and (ii) processing the mixture in agranulating apparatus until the granule has the desired particledistribution and degree of roundness (sphericity).

Numerous references describe processes for making enzyme-containinggranules by such a process. Such references include U.S. Pat. No.4,242,219, U.S. Pat. No. 4,740,469, WO 94/04665, U.S. Pat. No.4,940,665, EP 564476, EP 168526, U.S. Pat. No. 4,661,452, U.S. Pat. No.4,876,198, WO 94/16064 and U.S. Pat. No. 4,106,991.

Further, U.S. Pat. No. 5,494,600 and U.S. Pat. No. 5,318,903 describe aprocess comprising absorption of an enzyme into a porous hydrophobiccore (e.g. a porous hydrophobic silica core) followed by coating.

Although granulation techniques have improved, in order to take accountof increasing environmental concerns and heightened awareness in thefield of industrial hygiene, there remains a continuing need forenzyme-containing, granular compositions exhibiting even lower dustformation than presently available products.

An object of the present invention is to provide such enzyme-containinggranular compositions, and improved processes for producing suchcompositions.

SUMMARY OF THE INVENTION

It has surprisingly been found that extremely low tendency to dustformation by enzyme-containing granules is achievable when the granulesare based on suitably selected cores (particles), more specificallycores fulfilling, in particular, certain requirements with respect toliquid-absorption properties.

A first aspect of the present invention thus relates to anenzyme-containing granule comprising:

(a) an enzyme, and

(b) a core which intrinsically is capable of absorbing at least 5% byweight (w/w) of water (relative to the weight of the core).

In keeping with this first aspect of the invention, a further aspect ofthe invention relates to a process for producing enzyme-containinggranules from absorbent cores, the process comprising:

(a) contacting absorbent cores, capable of absorbing at least 5% w/w(based on the weight of the core) of water, with a liquid mediumcontaining an enzyme in dissolved and/or dispersed form, the amount ofthe liquid medium employed being such that substantially no attendantagglomeration of the resulting product occurs; and

(b) at least partially removing volatile components of the liquid mediumfrom the resulting product.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 describes the principle of a crushing test suitable for measuringthe strength of a core particle (or a finished enzyme-containinggranule) in the context of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Cores

Cores forming the basis of enzyme-containing granules of the invention(and in the context of processes according to the invention for thepreparation of such granules) are cores which, in the absence of ahydrophobicity-reducing substance or surface-tension-reducing substance(such as a surfactant) and of constituents of the enzyme-containinggranules (notably the enzyme or enzymes in question) other than those ofthe core itself, have an intrinsic (i.e. inherent, innate or “native”)ability to absorb at least 5% w/w of water when brought into contactwith water (i.e. essentially pure, liquid water).

Cores of relevance in the context of the invention are preferablycapable of absorbing at least 10% w/w (based on core weight) of water,more preferably at least 15% w/w and still more preferably at least 20%w/w. Particularly preferable cores are cores capable of absorbing atleast 30% w/w of water, such as cores capable of absorbing at least 33%w/w. Certain preferred types of cores have an even greaterwater-absorption capacity (e.g. about 40% w/w or more of water).

The ability of a particular type of core to absorb water may suitably bedetermined, for example, by spraying a measured amount of water onto ameasured amount of cores with mixing in a mixer [e.g. as described inExample 1 herein (vide infra)] and observing the appearance andbehaviour of the cores in the course of spraying; ambient conditions(ambient temperature, pressure etc.) are generally suitable. In general,as long as the amount of cores in question is able to absorb water thereis essentially no agglomeration of the wetted cores (i.e. agglomerationto form lumps or the like) and it is possible to remove the resultingindividual particles and dry them without significantly changing theoverall particle size distribution.

In the context of the invention, a quoted weight percentage of waterwhich a given type of core is capable of absorbing is thus a“macroscopic”, overall value determined using a relatively large amount(and thereby a large number) of cores (particles) of the given type,such as a multi-kilogram quantity (e.g. an amount of about 5, 10, 15 ormore kilograms of the given type of core).

If appropriate, smaller quantities of cores may, however, be employedfor the determination of water-absorption capacity, provided (i) thenumber of individual core particles in the sample employed issufficiently high to be adequately representative of the bulk cores, and(ii) the sample employed is sufficiently large to permit satisfactorilygradual addition of water (preferably by spraying), with adequatemixing, while observing the appearance/behaviour of the cores withrespect to surface wetness and tendency to agglomerate.

With a poorly absorbing or non-absorbing core material, agglomeration ofthe granules will normally occur upon introduction of only a smallamount of water, and consequently it will generally not be possible tomaintain the initial overall particle size distribution.

An example of a poorly absorbing core material is a conventionalsugar/starch-based particle of the so-called nonpareil type. In workingexamples herein (vide infra) it is shown, inter alia, that a typicalnonpareil core product is able to absorb less than 4% of water by weight(relative to the core).

Preferred embodiments of enzyme-containing granules of the invention aregranules wherein at least part of the total amount of enzyme present inthe granule is absorbed within the core; such granules will often begranules wherein at least part of the total amount of enzyme present inthe granule has been absorbed into the core via contact of the outersurface of the core with a liquid medium containing the enzyme inquestion.

Such embodiments appear [cf. the working examples herein (vide infra)]to be advantageous in relation to achieving improvement in a number ofcharacteristics or properties of enzyme-containing granules of theinvention relative to enzyme-containing granules of known types; thesecharacteristics or properties include:

tendency of the granules to form dust (particularly enzyme-containingdust);

enzymatic activity yield of the granules (i.e. degree of retention, in abatch of product granules, of the total original enzymatic activity ofthe enzyme preparation used to prepare the batch);

degree of retention of enzymatic activity during storage of the granulesunder various conditions; and

degree of retention of enzymatic activity following aggressivetreatments such as exposure to high temperatures and/or high humidity.

Moreover, without being bound to any theory it is believed [as alsomentioned below (see Example 11 herein)] that the presence, within thecore, of absorbed enzyme enhances the adherence of enzyme deposited onthe outer surface of an absorbent core of the type of relevance in thecontext of the invention, leading to corresponding reduction in thetendency to dust formation by such a product.

In relation to embodiments of enzyme-containing granules of theinvention wherein a part of the total enzyme content is absorbed withinthe core, valuable embodiments include those wherein at least 10% w/w,such as at least 25% w/w, e.g. at least 40% w/w of the total enzymecontent (calculated as active enzyme protein) is present as enzymeabsorbed within the core.

Particularly valuable embodiments include those wherein at least 90% w/wof the total enzyme content of the granule is present as enzyme absorbedwithin the core, and it is possible (employing, e.g., a processaccording to the invention) to obtain enzyme-containing granules of theinvention in which essentially all (i.e. essentially 100% w/w) of theenzyme content of the granule is present as enzyme absorbed within thecore.

In may be mentioned this connection that for a given distribution of theenzyme content of granules of the invention between (i) core-absorbedenzyme and (ii) enzyme present on the surface of the core or in/on oneor more coating layers, the absolute amount of enzyme protein which ispresent as enzyme absorbed within the core will, of course, depend onthe total amount of enzyme present in the granule.

Cores forming the basis of enzyme-containing granules of the invention,and cores employed in the process according to the invention, arepreferably shaped so that the ratio between the largest and the smallestdiameter thereof is less than 3; granules of the invention—whetheruncoated or coated (vide infra)—are likewise preferably shaped so thatthe ratio between the largest and the smallest diameter thereof is lessthan 3. For both cores and enzyme-containing granules, the latter ratiois preferably less than 2, more preferably ≦1.5 (i.e. between 1 and1.5), and it is particularly preferred that the ratio in question is atmost 1.2.

In should be noted that in the context of the present invention, valuesof the above-mentioned ratio between the largest and the smallestdiameters of cores or granules as recited herein are normally determinedas the mean value of the ratio in question for a representative numberof particles taken at random from a sample of the cores or granules inquestion. For the majority of preferred types/shapes of cores orgranules in the context of the invention, measurement of the ratio inquestion (e.g. by microscopy) for each of 20 or more particles taken atrandom from a sample of cores or granules, respectively, provides areliable basis for calculating a satisfactorily reproducible mean value.

For most purposes, cores forming the basis of enzyme-containing granulesof the invention, cores employed in the process according to theinvention, as well as enzyme-containing granules of the invention perse, are substantially spherical, i.e. such that the ratio in question isabout 1 (in that for a strictly spherical particle, the ratio inquestion is of course 1.0).

The ratio between the largest and the smallest diameter of a core or agranule may in general be taken as the ratio between the largest and thesmallest dimension (linear dimension) of the core or the granule,respectively, in a direction passing substantially through the center ofthe particle.

With regard to the term “center of the particle” it will be apparentthat particles (cores or granules) which are substantially axiallysymmetric, such as substantially spherical or ellipsoidal particles,will have a geometrically rather well-defined center. In such cases theterm “center of the particle” may be interpreted in a geometric manner.

Particles of more irregular shape will, however, in general not have ageometrically definable center, and in such cases the “center of theparticle” may [in that irregularly shaped particles (cores, granules)are—for most purposes—generally not preferred types in the context ofthe invention] be understood to be the center of gravity of the particlein question.

It is further preferred that cores forming the basis ofenzyme-containing granules of the invention, cores employed in theprocess according to the invention, as well as enzyme-containinggranules of the invention per se [whether the granules are uncoated orcoated (vide infra)] have a substantially smooth surface, i.e. a surfacewhich is essentially free of protuberances, spikes, secondary particles,irregularities, cavities, craters, indentations, pits and the like.

The degree of “smoothness” of cores or granules in the context of theinvention will, in general, be based on an overall assessment of thesurface characteristics of a representative sample of bulk cores orgranules, respectively. Particularly in the context of particles (coresor granules) which are substantially spherical, the term “smooth” mayfurther be taken to indicate that the mean value, for a representativenumber (e.g. ≧20 particles), of the ratio between the largest and thesmallest linear dimension of the particles, in a direction passingthrough the center of the particle, and measured on any given partialsegment of the outer surface of the particle, is less than 1.15,preferably less than 1.10, and more preferably less than 1.05.

Preferred types of cores in the context of the invention include corescomprising starch and/or modified starch, notably cores containing atotal of at least 25% w/w (based on total core weight), such as at least50% w/w, of starch and/or modified starch.

Enzyme-containing granules based on cores comprising a total of at least75% w/w of starch and/or modified starch, e.g. a total of at least 80%w/w, such as a total of at least 85% w/w (based on total core weight) ofstarch and/or modified starch, appear, in general, to possessparticularly advantageous properties (e.g. properties such as lowtendency to dust formation, heightened retention of enzyme activity,etc, as discussed earlier, above).

Highly preferred cores of this type are cores containing a total of atleast 90% w/w, particularly at least 95% w/w (based on total coreweight), of starch and/or modified starch, especially cores consistingessentially exclusively of (i.e. containing a total of essentially 100%w/w of) starch and/or modified starch.

It will be apparent that the above-mentioned total weight percentages (%w/w) of starch and/or modified starch are expressed as percentages ofthe weight of the core(s) per se, [i.e. the cores in their “native” orinnate state, not including the enzyme and/or any other components whichenter or adhere to the core(s) in the course of preparation ofenzyme-containing granules in accordance with the invention].

Starches (naturally occurring starches) from a wide variety of plantsources appear to be suitable in the context of the invention (either asstarches per se, or as the starting point for modified starches), andrelevant starches include starch from: cassava [notably from bittercassava (Manihot esculenta) or sweet cassava (Manihot dulcis)];sago-palm (Metroxylon spp., such as M. sagu); potato (Solanumtuberosum); rice (Oryza spp.); corn (maize, Zea mays); wheat (Triticumspp.); barley (Hordeum spp., such as H. vulgare) sweet potato (Ipomoeabatatas); sorghum (Sorghum spp.); and yam (Dioscorea spp.).

Other types of starch of potential value in the context of the inventioninclude starch from: rye (Secal cereals); oat (Avena spp., such as A.sativa); millet (e.g. from species of Digitaria, Panicum, Paspalum,Pennisetum or Setaria); buckwheat (Fagopyrum spp., such as F.esculentum); waxy maize; other cereals; arrowroot (e.g. Marantaarundinacea); taro (Colocasia spp., such as C. antiquorum or C.esculenta); tannia (Xanthosoma sagittifolium); Amaranthus spp.; andChenopodium spp.

Cassava starch is among preferred starches in the context of theinvention; in this connection it may be mentioned that cassava andcassava starch are known under various synonyms, including tapioca,manioc, mandioca and manihot.

As is well known, starches consist, in general, essentially ofmacromolecular polymers composed of α-D-glucopyranose units. Linear orsubstantially linear polymeric forms in which these units are linked byα-D-(1→4) linkages are known as “amylose”. Branched polymeric formscontaining α-D-glucopyranose units linked by both α-D-(1→4) andα-D-(1→6) linkages are known as “amylopectin”, the α-D-(1→6) linkagesaccounting typically for about 5-6% of the glycosidic linkages therein.

In this connection, starches from different vegetable sources containdifferent proportions of amylose and amylopection. Thus, for example,starch from potatoes typically contains ca. 20% w/w of amylose and ca.80% w/w of amylopectin, whereas so-called “waxy maize starch” generallycontains ≦2% w/w of amylose and ≧98% J/w of amylopectin.

In relation hereto it may be mentioned that a considerable amount ofeffort has been expended with a view to obtaining strains (e.g.genetically manipulated strains) of starch-producing plants which canproduce starches having an altered amylose/amylopectin balance (ratio)relative to that of starch produced by the plant as it occurs in nature.

Starches having widely differing proportions of amylose and amylopectin,respectively, are believed to be of value in the context of the presentinvention. Thus, high-amylose starches, high-amylopectin starches, andstarches having intermediate amylose/amylopectin ratios—includingstarches from genetically modified plant sources—are all of relevance inthe context of the invention, either as starches per se or as sources ofmodified starches.

As employed in the context of the present invention, the term “modifiedstarch” denotes a starch (native starch) which has undergone some kindof at least partial chemical modification, enzymatic modification,and/or physical or physicochemical modification, and which—ingeneral—exhibits altered properties relative to the “parent” starch.

Relevant chemical modifications include, but are not limited to:esterification of hydroxy groups (achieved, e.g., via acetyl-ation);etherification of hydroxy groups; oxidation (achieved, e.g., viareaction with chlorine or hypochlorite); and cross-linking (achieved,e.g., by reaction with formaldehyde or epichlorohydrin).

Etherified starches (e.g. carboxymethyl-starches orhydroxyalkyl-starches) and/or esterified starches can serve, inter alia,as binders (vide infra) in cores of relevance in the context of theinvention.

Relevant enzymatic modifications include, for example, treatment with astarch-degrading or starch-modifying enzyme, e.g. an amylase, such as anα-amylase or glucoamylase. In this connection, a particularlyinteresting possibility is modification of the absorption properties(and possibly other properties) of existing starch-containing cores,including cores consisting predominantly or essentially exclusively ofstarch and/or partly gelatinized starch (vide infra), by controlledtreatment thereof with a starch-degrading enzyme so as to modify(normally increase), the porosity/absorption capacity of cores [forexample via the creation of new pores, and/or via increase in the sizeand/or number and/or extent (“depth”) of existing pores therein].

Relevant physical or physicochemical modifications include, inparticular, so-called gelatinization. The term “gelatinized”, in thecontext of starch, is used herein in accordance with usage in the art(see, e.g., A. Xu and P. A. Seib, Cereal Chem. 70 (1993), pp. 463-70).

When a core comprising a porous aggregate of starch grains is partlygelatinized (e.g. by heating under water vapour pressure), it isbelieved that the starch grains situated at or near the outer surface ofthe core undergo a process wherein the amylose and/or amylopectin in thegrains in question forms an internal network/structure which results ingreater elasticity and physical strength of the outer part of the core.

The degree of gelatinization may suitably be determined usingdifferential scanning calorimetry (DSC) as described by A. Xu and P. A.Seib (loc cit.) (see Example 21 herein for further details).

In a farther aspect of the invention, very useful cores of the type(discussed above) comprising starch and/or modified starch are coreswhich comprise partly gelatinized starch; preferred cores of this typeinclude cores consisting essentially exclusively of partly gelatinizedstarch.

In such cores it is generally desirable that the degree ofgelatinization (vide infra) of the starch is greater than 0.5%, such asat least 2%, and at most 95%. For many types of starch, a preferredrange of degree of gelatinization in this connection is from 10% to 60%gelatinization.

Starch-containing cores with very low degrees of starch gelatinizationappear to have lower physical strength than corresponding cores withhigh degrees of starch gelatinization. On the other hand, thewater-absorption ability of starch-containing cores with high degrees ofstarch gelatinization appears to be lower than for cores with lowerdegrees of starch gelatinization.

Particularly—but not exclusively—in the case of cores consistingpredominantly, or essentially solely, of partly gelatinized starch,degrees of starch gelatinization in the range of 30-60%, such as in therange of 30-50%, appear to be associated, inter alia, with a combinationof very satisfactory absorption capacity, a high degree of sphericityand surface smoothness, and satisfactorily high physical strength(resistance to crushing). As is apparent from the working examplesherein (vide infra), starch-based cores of this type can be supplied byexisting suppliers, and have been found to exhibit a very desirablecombination of properties in the context of numerous aspects within thescope of the present invention.

In connection with the use, in particular, of starches as core materialsin the context of the invention, it is contemplated that as analternative to substantially homogeneous, starch-based core particles itwill be possible to employ core particles which comprise a layer ofabsorbent starch (e.g. cassava starch or rice starch) deposited on aninner carrier material (e.g. an insoluble silicate, carbonate or thelike) which may or may not itself possess the water-absorptionproperties which are otherwise characteristic of cores in the context ofthe invention.

In this connection, further types of absorbent cores which appear to besuitable in the context of the invention include cores comprising anon-hydrophobic silicate or siliceous material as an absorbent material.Examples of such materials which may be prepared in, or are availablein, granular form are bentonite, fuller's earth (both of which consistpredominantly of the smectite mineral montmorillonite), diatomaceousearths (infusorial earths, e.g. kieselguhr, tripolite, tripoli ordiatomite) and other smectite minerals (such as beidellite, nontronite,saponite, sauconite or hectorite).

Cores forming the basis of enzyme-containing granules according to theinvention, or employed in the process of the invention, may—whereappropriate and relevant—comprise one or more materials (additives oradjuvants) such as binders, fillers, plasticizers, fibrous materialsand/or so-called “superabsorbents”.

Binders: When incorporated in cores in the context of the invention,binder(s) will suitably be present in amounts constituting up to about20% of the total weight of the core. Appropriate binders will generallybe binders which are conventionally used in the field of granulation andwhich have a high melting point, or do not melt, and are of a non-waxynature. Such binders may be low or high molecular weight binders,including water-soluble binders and water-based emulsion binders.Included in this connection are carbohydrate-type binders ranging fromsubstances of the monosaccharide type to substances of thepolysaccharide type, as well as derivatives thereof.Oligosaccharide-type binders, e.g. certain dextrins, are often wellsuited.

Examples of binders of the polysaccharide derivative type include starchderivatives (some types of which have already been mentioned above inconnection with modified starches), such as starch esters (e.g. starchacetate), starch ethers (such as carboxymethyl-starch orhydroxyalkyl-starches, e.g. hydroxymethyl-, hydroxyethyl- orhydroxypropyl-starch) and cellulose derivatives, such asmethylhydroxypropyl-cellulose, hydroxypropyl-cellulose,methyl-cellulose, carboxymethyl-cellulose (CMC) as well as CMC sodiumsalt.

Further examples of relevant binders include polyacrylates,polymethacrylates, acrylic acid/maleic acid copolymers andvinyl-group-containing compounds, such as polyvinyl alcohol, hydrolysedpolyvinyl acetate and polyvinylpyrrolidone. Fillers: Fillers appropriatefor incorporation in cores in the context of the present inventioninclude inert materials used to add bulk and reduce cost, or used forthe purpose of adjusting the intended enzyme activity in the finishedgranulate. Examples of such fillers include, but are not limited to,water-soluble substances such as urea, various salts (such as sodiumchloride, ammonium sulphate or sodium sulphate) and sugars, andwater-dispersible agents such as clays, talc, silicates or starches.

Plasticizers: In certain types of cores of relevance in the context ofthe invention, plasticizer(s) may suitably be present in amountsconstituting up to about 10% of the total weight of the core.Plasticizers serve generally to reduce brittleness and/or enhancedeformability, and will typically be low molecular weight organiccompounds of low volatility [such as polyols (e.g. glycols such asethylene glycol), urea and phthalate esters (such as dibutyl or dimethylphthalate)]. Water may in some cases serve as a plasticizer.

Fibrous materials: When incorporated in cores in the context of theinvention, fibrous material(s) will suitably be present in amountsconstituting up to about 30% of the total weight of the core, preferablybetween 5 and 15%. Suitable fibrous materials include materials whichhave high tensile strength and are in the form of fine filaments havinga diameter of 1-50 μm and a length equal to at least four diameters.Typical fibrous materials include, but are not limited to: cellulosefibres, glass fibres, metal fibres, rubber fibres, azlon fibres(manufactured from naturally occurring proteins from corn, peanuts andmilk) and synthetic polymer fibres (such as fibres of Rayon™, Nylon™,polyester, polyolefin, Saran™, Spandex™ and vinal™. Cellulose fibres arevery suitable fibres in this connection, and will often suitably have anaverage fibre length in the range of 150-300 μm and a diameter in therange of about 20-40 μm.

Superabsorbents: Certain types of—in particular—macro-molecularsubstances possess the ability to absorb many times their own weight ofwater or certain aqueous media. Substances of this type [which includevarious types of synthetic polymers as well as substances derived frompolymers of natural origin, and which have found applications, forexample, as body fluid absorbents (e.g. in wound dressings, diapers,sanitary towels and the like)] are sometimes referred to as“superabsorbents”, and such substances may be present as a component ofcores which form the basis for enzyme-containing granules of theinvention.

One group of interesting superabsorbent materials, described in WO96/03440, comprises readily biodegradable substances derived—via anenzymatic process—from certain naturally occurring phenolicpolysaccharides, such as phenolic pectins, and such substances are wellsuited as water-absorbent components in cores of relevance in thecontext of the invention.

At this point it is appropriate to mention another class of absorbentcores which are well suited as the basis for enzyme-containing granulesof the invention, namely “placebo” (enzyme-free) cores prepared inaccordance with the general methodology of U.S. Pat. No. 4,106,991 butwithout the inclusion of an enzyme. U.S. Pat. No. 4,106,991 describesthe production of so-called “T-granulates” comprising—in addition toenzyme—2-40% w/w of fibrous cellulose, together with binder (e.g. one ofthose mentioned above in the context of binders for cores or relevancein the context of the invention), filler (typically a salt such assodium sulphate or sodium chloride, optionally together with a minorproportion of a whitener such as titanium dioxide or kaolin) and aliquid-phase granulating agent (water and/or a waxy substance such as apolyglycol, fatty alcohol, ethoxylated fatty alcohol or the like). Bypreparing granulates in accordance with U.S. Pat. No. 4,106,991 (whichemploys drum granulation of a mixture of enzyme, fibrous cellulose,binder, filler, and liquid-phase granulating agent), but withoutincorporation of enzyme, absorbent cores which are well suited as coresin the context of the present invention may be prepared. Cores of thistype, which may appropriately be termed “placebo T” cores, may also beprepared using “filler” materials other than salts (such as powderedstarch, e g. powdered rice starch).

As already indicated to some extent above, it is preferable that coreparticles forming the basis of enzyme-containing granules of theinvention have relatively high physical strength. In the context of theinvention the strength of a substantially spherical core particle maysuitably be determined as the ratio between the force required toinitiate crushing of the particle under the test conditions as specifiedbelow (vide infra), and the square of the core diameter (i.e. regardingthe particle as being substantially spherical).

It is preferable that the mean value of this ratio, determined for arepresentative number of cores (suitably >20 cores) taken at random froma bulk quantity of cores, is greater than 400 g/mm², more preferablygreater than 600 g/mm², such as greater than 800 g/mm². It is especiallydesirable that the ratio in question is greater than 1000 g/mm², morepreferably greater than 1200 g/mm², in particular greater than 1400g/mm², and most preferably greater than 1600 g/mm².

The principle of a crushing test suitable for measuring the strength ofa core particle (or a finished enzyme-containing granule) in the contextof the present invention is illustrated in FIG. 1 herein (vide infra).The test is performed as follows:

1) A particle of core material is placed between an aluminium plate anda Plexiglas™ [transparent poly(methyl methacrylate)-type polymer] plate(each plate measuring 218 mm×40 mm, thickness 3.2 mm) as shown in FIG.1; the Plexiglas™ plate is strengthened by two aluminium U-profilesattached to the edges of the plate [as indicated in the “front view”(end view) at the bottom of FIG. 1] and extending along the fall lengthof the plate;

2) increasing loads (circular/cylindrical weights) are placedsuccessively on the end of the Plexiglas™ plate, closest to the core, asshown in FIG. 1, such that the center of mass of the weight ispositioned in the middle of the width of the plate, and 20 mm from theend thereof; at the same time, the particle is observed through thetransparent plate by means of a microscope;

3) the loading (in grams) at which crushing of the particle begins (asassessed visually) is measured, and is divided by the square of thediameter of the core particle (in mm²) to obtain the strength of thecore particle.

Alternatively, the physical strength of particles of core material (orof granules according to the invention) can be measured according toeither the Heubach method or the Novo Nordisk attrition method, both ofwhich give a measure of the dust-formation tendency of particles;protocols for the latter two methods (EAL-SM-0289.01/01 and AF 225/2-GB,respectively) are obtainable on request from Novo Nordisk A/S,Bagsvaerd, Denmark. In both of the latter methods a bed of particles issubjected to the action of rolling steel balls, with simultaneoussuction of air through the bed to collect dust and fragments createdduring the process.

For numerous applications of enzyme-containing granules of theinvention, the mean particle size of the granules (and in many cases,correspondingly, of the core particles therein) will suitably be in therange from 50 to 4000 μm, such as 200-2000 μm (e.g. in the range of200-1000 μm). The optimal mean core particle size will generally dependon the intended use of the final enzyme-containing granulate.

Embodiments of enzyme-containing granulates of the invention are wellsuited for use, for example, in detergents, in animal feed compositions,in baking and in the treatment of textiles. By way of example, fordetergent applications the preferred mean granule particle size (and, inmany cases, the corresponding mean core particle size) will often be inthe range of 250-2000 um (such as 300-2000 μm), whereas for bakingapplications the preferred mean core particle size will often be in therange of 50-200 μm. Granules (and, correspondingly, often cores) of asize greater than 4000 μm, such as particles of size of the order of10000 μm, may be appropriate for certain applications (e.g. in thetreatment of textiles).

The overall particle size distribution is preferably relatively narrow,e.g. such that for at least 90%, more preferably 95%, of the particlesin a given sample the ratio between the largest and the smallestparticle size is less than 4:1, preferably less than 3:1, morepreferably less than 2:1, and most preferably less than 1.5:1.

Granular core particles suitable as the basis for enzyme-containinggranules in accordance with the present invention may be prepared, e.g.,by conventional granulation methods such as tumbling, rolling,pelletization, extrudation/-spheroidization, and/or mechanical agitationof the starting material, e.g. starting material comprising starch or asilicate/siliceous material. Examples of suitable absorbent cores(“placebo T” cores) which may be prepared by drum granulation (in themanner disclosed in U.S. Pat. No. 4,106,991) are described above (seealso Examples 8 and 13 herein).

Coating layers

The granules of the present invention may comprise one, two or morecoating layers. Such coating layers may, for example, be one or moreintermediate coating layers, or one or more outside coating layers, or acombination thereof.

Coating layers may perform any of a number of functions in a granulecomposition, depending on the intended use of the enzyme granule. Thus,for example, a coating may achieve one or more of the following effects:

(i) further reduction of the dust-formation tendency of an uncoatedgranule according to the invention;

(ii) protection of enzyme(s) in the granule against oxidation bybleaching substances/systems (e.g. perborates, percarbonates, organicperacids and the like);

(iii) dissolution at a desired rate upon introduction of the granuleinto a liquid medium (such as an aqueous medium);

(iv) provision of a barrier against ambient moisture in order to enhancethe storage stability of the enzyme and reduce the possibility ofmicrobial growth within the granule.

In appropriate embodiments of granules according to the presentinvention, the coating layer may be composed as described in U.S. Pat.No. 4,106,991 [e.g. with a waxy material such as polyethylene glycol(PEG), optionally followed by powdering with a whitener such as titaniumdioxide].

A given coating layer may contribute from 0.5% to as much as 50% byweight of the finished granule.

Coating layers in/on granules of the present invention may furthercomprise one or more of the following: anti-oxidants, chlorinescavengers, plasticizers, pigments, lubricants (such as surfactants orantistatic agents) and additional enzymes.

Plasticizers useful in coating layers in the context of the presentinvention include, for example: polyols such as sugars, sugar alcohols,or polyethylene glycols (PEGs) having a molecular weight less than 1000;urea, phthalate esters such as dibutyl or dimethyl phthalate; and water.

Suitable pigments include, but are not limited to, finely dividedwhiteners, such as titanium dioxide or kaolin, coloured pigments, watersoluble colorants, as well as combinations of one or more pigments andwater soluble colorants.

As used in the present context, the term “lubricant” refers to any agentwhich reduces surface friction, lubricates the surface of the granule,decreases tendency to build-up of static electricity, and/or reducesfriability of the granules. Lubricants can also play a related role inimproving the coating process, by reducing the tackiness of binders inthe coating. Thus, lubricants can serve as anti-agglomeration agents andwetting agents.

Examples of suitable lubricants are polyethylene glycols (PEGs) andethoxylated fatty alcohols.

As already mentioned, the present invention also relates to a processfor producing enzyme-containing granules from absorbent cores, theprocess comprising:

(a) contacting absorbent cores, capable of absorbing at least 5% w/w(based on the weight of the core) of water, with a liquid mediumcontaining an enzyme in dissolved and/or dispersed form, the amount ofthe liquid medium employed being such that substantially no attendantagglomeration of the resulting product occurs; and

(b) at least partially removing volatile components of the liquid mediumfrom the resulting product.

Preferred characteristics of cores suitable for use in the process ofthe invention are those already discussed above in connection withenzyme-containing granules of the invention.

In the process of the invention, the contacting of the absorbent coreswith a liquid medium (such as an aqueous medium) comprising dissolvedand/or dispersed enzyme is suitably carried out, for example, byspraying the cores with the solution/dispersion under mixing conditions,or by applying (e.g. by spraying) the enzyme solution/dispersion tocores which are fluidized (e.g. in a fluid-bed apparatus), or by acombination of both techniques.

Contacting of cores with enzyme via a mixing technique is generally wellsuited in the context of the invention, since it facilitates—whenappropriate—addition, in step (a) of the process of the invention, ofthe enzyme-containing solution or dispersion to the cores in an amount,and for a period of time, which is sufficient to wholly (or partly)exploit the absorption capacity of the cores, but which does not leaveany significant amount of free liquid phase (i.e. enzyme-containingsolution or dispersion) on or between the resulting individualparticles, i.e. such that there is insufficient free (unabsorbed) liquidphase to cause agglomeration of the particles to occur. If surplus(unabsorbed) liquid phase is present there is a risk of agglomerationoccurring, with attendant unwanted formation of lumps in the bulkgranulate.

Conventional mixing equipment can satisfactorily be used to mix thecores with the enzyme-containing liquid medium. The mixing equipment canbe a batch mixer or a continuous mixer, such as a convective mixer [see,e.g., Harnby et al., Mixing in the Process Industries, pp. 39-53 (ISBN0-408-11574-2)]. Non-convective mixing equipment, e.g. rotating drummixers or so-called pan-granulators, may also be employed.

As already indicated, conditions whereby the cores are fluidized (suchas in a fluid-bed apparatus or other form of fluidizing equipment, suchas a Hüttlin-type fluidizer) may also be employed when contacting thecores and the enzyme-containing liquid medium. For a description ofsuitable fluid-bed equipment, see, e.g., Hamby et al., Mixing in theProcess Industries, pp. 54-77 (ISBN 0-408-11574-2).

In general, it is advantageous that the enzyme-containing liquid mediumemployed in the process of the invention contains dissolved enzyme. Thiswill normally be the case when working with aqueous media. It is furtherdesirable that the process is carried out under conditions such thatabsorption of the liquid phase (which will often contain dissolvedenzyme) of the liquid medium by the cores takes place to some extent instep (a) of the process, often preferably to an extent such thatessentially complete absorption of the liquid phase by the cores takesplace before taking any measures to remove volatile components of theliquid medium from the resulting product.

In this connection, the process of the invention may thus be performed,for example, under conditions whereby essentially no removal, or atleast very little removal, of volatile components takes place during thecontacting phase. When using a mixer set-up in the contacting step, thismay generally be achieved by simply ensuring that temperature in themixer is not too high (e.g. such that the temperature is ambienttemperature or below); when using fluidized conditions in the contactingstep, this condition may generally be met by employing fluidizing air ofsufficiently low temperature (e.g. a temperature below 30° C., such asambient temperature or below).

Volatile components may subsequently be removed, for example, in amixer, under mixing conditions (e.g. by applying heat and/or reducedpressure) or under fluidized conditions, e.g. in a fluid-bed apparatus(for example by the application of suitably hot fluidizing air). Thetemperatures employed should, of course, be such that no significantloss of enzyme activity of the product granules occurs.

Alternatively, some degree of evaporation of volatile components may beallowed to take place simultaneously with the performance of thecontacting step. Thus, for example, when employing a mixer set-up in thecontacting step, the mixer may be heated to a moderately elevatedtemperature in order to cause some evaporation of volatiles during thecontacting stage; when employing fluidized conditions in the contactingstep, the fluidizing air itself may be heated to a moderately elevatedtemperature. As before, the temperatures employed should, of course, besuch that no significant loss of enzyme activity of the product granulesoccurs.

When drying product granules, they may suitably be retained in theprocess apparatus for a period of time sufficient to reduce the moisturecontent (in the case of products prepared using an aqueous,enzyme-containing medium) to a level below 10% w/w free moisture,preferably below 5% w/w free moisture.

As already discussed (vide supra), cores employed in the context of theinvention are intrinsically able to absorb suitable amounts of water(and thereby of aqueous media), and consequently there is no need toinclude a surfactant in the enzyme-containing liquid medium in order forthe cores to be able to absorb a sufficient amount of a hydrophilicpolypeptide such as an enzyme.

This is particularly advantageous with respect to the production ofenzyme-containing granulates for use in industries such as the bakingindustry or the animal feed industry, where the presence of surfactantin the granules is generally undesirable.

Nevertheless, in certain situations, such as when a product of higherenzymatic activity than usual is required (e.g. for use in the detergentindustry), it may be advantageous, at least for certain types ofabsorbent core, to include an appropriate amount of surfactant in theenzyme-containing liquid medium for the purpose of enhancing and/oraccelerating absorption of enzyme by the core. Surfactants suitable forthis purpose include numerous types of cationic, anionic, non-ionic orzwitterionic surfactants, and suitable examples hereof are mentionedbelow, in connection with the discussion of detergent compositions (videinfra).

In order to promote formation of a dense, compact (smooth/regular)granule surface after contacting the cores with the enzyme-containingliquid phase, the presence in the mixer of a rapidly rotatinggranulating device (“choppers”) is preferable. Reference may be made toU.S. Pat. No. 4,106,991 for further details.

Another way in which to promote the formation of a dense, compact(smooth/regular) surface after contacting the cores with the liquidmedium is to treat the moist granulate in a Marumerizer™. Reference maybe made to U.S. Pat. No. 4,106,991 for further details.

When an aqueous, enzyme-containing solution/dispersion is employed inaccordance with the invention, the solution (i.e. the liquid phase ofthe aqueous medium) will normally preferably have a dry matter contentof from 2% w/w to 50% w/w [dry matter consisting of enzyme protein(s),possibly together with other organic and inorganic materials]. Whendispersed enzyme is present, the solution/dispersion will suitably havea dry matter content of from 10% w/w to 70% w/w [including dry matteroriginating from both dissolved and dispersed (undissolved) material].

The term “dispersion” as used in this connection designates a systemcontaining solid particles, at least some of which comprise or consistof enzyme, of a size from the colloidal size range and upwards, andwhich are suspended, slurried or otherwise distributed in a liquid phase(such as an aqueous phase). The term “dispersion” in the context of theinvention thus embraces, inter alia, suspensions and slurries.

In a preferred aspect of the process of the invention, dispersed enzymepresent in the liquid medium employed in step (a) comprises enzyme incrystalline form.

In the process of the invention it is preferable that theenzyme-containing liquid medium (solution or solution/-dispersion) isadded to the particulate cores in a weight ratio (liquid medium: cores)of at least 0.05:1, more preferably at least 0. 1:1, such as at least0.15:1, for example at least 0.2:1, and often at least 0.5:1. The ratioemployed will, in general, depend on the absorption capacity of thecores, and on the required strength of the final enzyme-containinggranules.

One, two or more coating layers may be applied to the dried or partlydried, enzyme-containing granules by conventional methods, such as bypan-coating, mixer-coating, and/or fluid-bed coating. Suitablecoatings/coating components include those already discussed above inconnection with enzyme-containing granules of the invention.

Enzymes

Any enzyme or combination of different enzymes may be employed in thecontext of the present invention. Accordingly, when reference is made to“an enzyme” this will in general be understood include combinations ofone or more enzymes.

The enzyme classification employed in the present specification withclaims is in accordance with Recommendations (1992) of the NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology, Academic Press, Inc., 1992.

It is to be understood that enzyme variants (produced, for example, byrecombinant techniques) are included within the meaning of the term“enzyme”. Examples of such enzyme variants are disclosed, e.g., in EP251,446 (Genencor), WO 91/00345 (Novo Nordisk A/S), EP 525,610 (Solvay)and WO 94/02618 (Gist-Brocades NV).

Types of enzymes which may appropriately be incorporated in granules ofthe invention include:

hydrolases [EC 3; such as lipases (EC 3.1.1.3) and other carboxylicester hydrolases (EC 3.1.1); phytases, e.g. 3-phytases (EC 3.1.3.8) and6-phytases (EC 3.1.3.26); α-amylases (EC 3.2.1.1) and other glycosidases(EC 3.2, which fall within a group denoted herein as “carbohydrases”);peptidases (EC 3.4, also known as proteases); and other carbonylhydrolases];

oxidoreductases [EC 1; such as peroxidases (EC 1.11.1), laccases (EC1.10.3.2) and glucose oxidases (EC 1.1.3.4)];

transferases (EC 2); isomerases (EC 5); and ligases (EC 6).

Examples of commercially available proteases (peptidases) includeEsperase™, Alcalase™, Neutrase™, Durazym™, Savinasen™, Pyrase™,Pancreatic Trypsin NOVO (PTN), Bio-Feed™ Pro and Clear-Lens™ Pro (allavailable from Novo Nordisk A/S, Bagsvaerd, Denmark).

Other commercially available proteases include Maxatase™, Maxacal™,Maxapem™, Opticlean™ and Purafect™ (available from GenencorInternational Inc. or Gist-Brocades).

Examples of commercially available lipases include Lipolase™, Lipolase™Ultra, Lipozyme™, Palatase™, Novozym™ 435 and Lecitase™ (all availablefrom Novo Nordisk A/S).

Other commercially available lipases include Lumafast™ (Pseudomonasmendocina lipase from Genencor International Inc.); Lipomax™ (Ps.pseudoalcaligenes lipase from Gist-Brocades/Genencor Int. Inc.; andBacillus sp. lipase from Solvay enzymes. Further lipases are availablefrom other suppliers.

In the present context, the term “carbohydrase” is used to denote notonly enzymes capable of breaking down carbohydrate chains (e.g.starches) of especially five- and six- membered ring structures (i.e.glycosidases, EC 3.2), but also enzymes capable of isomerizingcarbohydrates, e.g. six-membered ring structures such as D-glucose tofive- membered ring structures such as D-fructose.

Carbohydrases of relevance include the following (EC numbers inparentheses): α-amylases (3.2.1.1), β-amylases (3.2.1.2), glucan1,4-α-glucosidases (3.2.1.3), cellulases (3.2.1.4),endo-1,3(4)-β-glucanases (3.2.1.6), endo-1,4-β-xylanases (3.2.1.8),dextranases (3.2.1.11), chitinases (3.2.1.14), polygalacturonases(3.2.1.15), lysozymes (3.2.1.17), β-glucosidases (3.2.1.21),α-galactosidases (3.2.1.22), β-galactosidases (3.2.1.23),amylo-1,6-glucosidases (3.2.1.33), xylan 1,4-β-xylosidases (3.2.1.37),glucan endo-1,3-β-D-glucosidases (3.2.1.39), α-dextrinendo-1,6-α-glucosidases (3.2.1.41), sucrose α-glucosidases (3.2.1.48),glucan endo-1,3-α-glucosidases(3.2.1.59), glucan 1,4-β-glucosidases(3.2.1.74), glucan endo-1,6-α-glucosidases(3.2.1.75), arabinanendo-1,5-α-L-arabinosidases (3.2.1.99), lactases (3.2.1.108),chitosanases (3.2.1.132) and xylose isomerases (5.3.1.5).

Examples of commercially available carbohydrases include Alpha-Gal™,Bio-Feed™ Alpha, Bio-Feed™ Beta, Bio-Feed™ Plus, Bio-Feed™ Plus,Cellusoft™, Ceremyl™, Citrozym™, Denimax™, Dezyme™, Dextrozyme™,Finizym™, Fungamyl™, Gamanase™, Glucanex™, Lactozym™, Maltogenase™,Pentopan™, Pectinex™; Promozyme™, Pulpzyme™, Novamyl™, Termrnamyl™, AMG™(Amyloglucosidase Novo), Maltogenase™, Sweetzyme™ and Aquazym™ (allavailable from Novo Nordisk A/S). Further carbohydrases are availablefrom other suppliers.

Examples of commercially available oxidoreductases (EC 1) includeGluzyme™ (enzyme available from Novo Nordisk A/S). Furtheroxidoreductases are available from other suppliers.

Suitable transferases (EC 2) in the context of the invention aretransferases in any of the following sub-classes: transferasestransferring one-carbon groups (EC 2.1); transferases transferringaldehyde or ketone residues (EC 2.2); acyltransferases (EC 2.3);glycosyltransferases (EC 2.4); transferases transferring alkyl or arylgroups, other that methyl groups (EC 2.5); and transferases transferringnitrogeneous groups (EC 2.6).

A preferred type of transferase in the context of the invention is atransglutaminase (protein-glutamine γ-glutamyltransferase; EC 2.3.2.13).

Examples of transglutaminases are described in WO 96/06931 (Novo NordiskA/S).

The amount of enzyme to be incorporated in a granule of the inventionwill depend on the intended use of the granulate. For many applications,the enzyme content will be as high as possible or practicable.

The content of enzyme (calculated as pure enzyme protein) in a granuleof the invention will typically be in the range of from about 0.5% to20% by weight of the enzyme-containing granule.

When, for example, a protease (peptidase) is incorporated in granulesaccording to the invention, the enzyme activity (proteolytic activity)of the finished granules will typically be in the range of 1-20 KNPU/g.Likewise, in the case of, for example, α-amylases, an activity of 10-500KNU/g will be typical, whilst for lipases, an activity in the range of50-400 KLU/g will normally be suitable.

Other Adjunct Ingredients

Where appropriate, various additives (adjuncts) may be incorporatedtogether with the enzyme in a granule of the invention. Relevantadjuncts agents may include: metal compounds (e.g. salts and/orcomplexes of transition metals), solubilizers, activators,anti-oxidants, dyes, inhibitors, binders, fragrances, enzyme-protectingagents/scavengers, such as ammonium sulphate, ammonium citrate, urea,guanidine hydrochloride, guanidine. carbonate, guanidine sulphonate,thiourea dioxide, monoethanolamine, diethanolamine, triethanolamine,amino acids such as glycine, sodium glutamate and the like, proteinssuch as bovine seruim albumin, casein, surfactants, including anionicsurfactants, ampholytic surfactants, nonionic surfactants, cationicsurfactants and long-chain fatty acid salts, builders, alkalis orinorganic electrolytes, bleaching agents, blueing agents and fluorescentdyes, and caking inhibitors. Reference may be made to WO 92/00384 for adescription of appropriate surfactants.

Granules incorporating such adjuvants may be made by methods well knownto those skilled in the art of enzyme granulation, including fluidizedbed spray-coating, pan-coating and other techniques for building up agranule by adding consecutive layers on top of a starting core material.

In addition to the process, according to the invention, for theproduction of enzyme-containing granules as described above, the presentinvention further relates to enzyme-containing granules obtained by, orobtainable by, an embodiment of the process.

Applications of enzyme-containing granules of the invention

Granulates (enzyme-containing granules) according to the presentinvention can be used for a variety of industrial applications.Particulary interesting applications include their use in detergents, inanimal feed compositions, in products for the baking industry and intextile-treatment products, and the following list indicates types ofenzymes which are most typically of interest in each of these fields ofapplication:

Detergents: proteases, amylases (e.g. α-amylases), cellulases, lipases,oxidoreductases;

Baking products: amyloglucosidases (glucoamylases, glucan1,4-α-glucosidases), bacterial α-amylases, fungal α-amylases, maltogenicamylases, glucose oxidases, proteases, pentosanases;

Animal feed compositions: bacterial α-amylases, proteases, xylanases;phytases;

Textile-treatment products: cellulases, α-amylases.

In a further aspect, the invention thus relates to detergentcompositions (especially laundry and dishwashing detergent compositions)comprising enzyme-containing granules according to, or produced inaccordance with, the invention.

In yet another aspect, the invention encompasses animal feedcompositions comprising enzyme-containing granules according to, orproduced in accordance with, the invention.

A still further aspect relates to compositions for baking comprisingenzyme-containing granules according to, or produced in accordance with,the invention.

Moreover, the invention also relates to the use of enzyme-containinggranules according to, or produced in accordance with, the invention asan enzyme-containing component in:

a detergent composition, e.g. for laundry washing or dishwashing;

an animal feed composition;

a composition for baking; or

a composition for textile treatment (e.g. for colour clarification orfor dyeing).

Detergent Compositions

According to the invention, the enzyme-containing granules of theinvention may typically be a component of a detergent composition, e.g.,a laundry detergent composition or a dishwashing detergent composition.As such, they may be included in the detergent composition in the formof an uncoated granulate or coated granulate coated by methods known inthe art. Examples of waxy coating materials are poly(ethylene oxide)products (polyethyleneglycol, PEG) with mean molecular weights of 1000to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxideunits; ethoxylated fatty alcohols in which the alcohol contains from 12to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units;fatty alcohols; fatty acids; and mono- and di- and triglycerides offatty acids. Examples of film-forming coating materials suitable forapplication by fluid bed techniques are given in patent GB 1483591.

The detergent composition of the invention may be in any convenientform, e.g. as powder, granules or paste.

The detergent composition comprises one or more surfactants, each ofwhich may be anionic, nonionic, cationic, or amphoteric (zwitterionic).The detergent will usually contain 0-50% of anionic surfactant such aslinear alkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkylsulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS orAES), secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methylesters, alkyl- or alkenylsuccinic acid, or soap. It may also contain0-40% of nonionic surfactant such as alcohol ethoxylate (AEO or AE),alcohol propoxylate, carboxylated alcohol ethoxylates, nonylphenolethoxylate, alkylpolyglycoside, alkyldimethylamine oxide, ethoxylatedfatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxyalkyl fatty acid amide (e.g. as described in WO 92/06154).

In addition to the enzymes contained in the enzyme-containing granulesof the invention, the detergent composition may additionally compriseone or more other enzymes, such as a pullulanase, esterase, lipase,cutinase, protease, cellulase, amylase, peroxidase or oxidase (e.g. alaccase).

Normally the detergent contains 1-65% of a detergent builder, but somedishwashing detergents may contain even up to 90% of a detergentbuilder, or complexing agent such as zeolite, diphosphate, triphosphate,phosphonate, citrate, nitrilotriacetic acid (NTA),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTMPA), alkyl-or alkenylsuccinic acid, soluble silicates orlayered silicates (e.g. SKS-6 from Hoechst).

The detergent builders may be subdivided into phosphorus-containing andnon-phosphorous-containing types. Examples of phosphorus-containinginorganic alkaline detergent builders include the water-soluble salts,especially alkali metal pyrophosphates, orthophosphates, polyphosphatesand phosphonates. Examples of non-phosphorus-containing inorganicbuilders include water-soluble alkali metal carbonates, borates andsilicates as well as layered disilicates and the various types ofwater-insoluble crystalline or amorphous alumino silicates of whichzeolites is the best known representative.

Examples of suitable organic builders include alkali metal, ammonium orsubstituted ammonium salts of succinates, malonates, fatty acidmalonates, fatty acid sulphonates, carboxymethoxy succinates,polyacetates, carboxylates, polycarboxylates, aminopolycarboxylates andpolyacetyl carboxylates.

The detergent may also be unbuilt, i.e. essentially free of detergentbuilder.

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylatessuch as polyacrylates, polymaleates, maleic/acrylic acid copolymers andlauryl methacrylate/acrylic acid copolymers.

The detergent composition may contain bleaching agents of thechlorine/bromine-type or the oxygen-type. The bleaching agents may becoated or incapsulated. Examples of inorganic chlorine/bromine-typebleaches are lithium, sodium or calcium hypochlorite or hypobromite aswell as chlorinated trisodium phosphate. The bleaching system may alsocomprise a H₂O₂ source such as perborate or percarbonate which may becombined with a peracid-forming bleach activator such astetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS).

Examples of organic chlorine/bromine-type bleaches are heterocyclicN-bromo and N-chloro imides such as trichloroisocyanuric,tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids,and salts thereof with water solubilizing cations such as potassium andsodium. Hydantoin compounds are also suitable. The bleaching system mayalso comprise peroxyacids of, e.g., the amide, imide, or sulfone type.

In dishwashing detergents the oxygen bleaches are preferred, for examplein the form of an inorganic persalt, preferably with a bleach precursoror as a peroxy acid compound. Typical examples of suitable peroxy bleachcompounds are alkali metal perborates, both tetrahydrates andmonohydrates, alkali metal percarbonates, persilicates andperphosphates. Preferred activator materials are TAED or NOBS

The enzymes of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g. a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative such as, e.g., an aromatic borateester, and the composition may be formulated as described in, e.g., WO92/19709 and WO 92/19708. The enzymes of the invention may also bestabilized by adding reversible enzyme inhibitors, e.g., of the proteintype as described in EP 0 544 777 B1.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, deflocculantmaterial, foam boosters/foam depressors (in dishwashing detergents foamdepressors), suds suppressors, anti-corrosion agents, soil-suspendingagents, anti-soil-redeposition agents, dyes, dehydrating agents,bactericides, optical brighteners, or perfume.

The pH (measured in aqueous solution at use concentration) will usuallybe neutral or alkaline, e.g. in the range of 7-11.

Particular forms of laundry detergent compositions within the scope ofthe invention include:

1) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

Linear alkylbenzenesulfonate (calculated as acid)    7-12% Alcoholethoxysulfate (e.g. C₁₂₋₁₈ alcohol, 1-2 EO)    1-4% or alkyl sulfate(e.g. C₁₆₋₁₈) Alcohol ethoxylate (e.g. C₁₄₋₁₅ alcohol, 7 EO)    5-9%Sodinm carbonate (as Na₂CO₃)    14-20% Soluble silicate (as Na₂O,2SiO₂)   2-6% Zeolite (as NaAlSiO₄)    15-22% Sodium sulfate (as Na₂SO₄)   0-6% Sodium citrate/citric acid (as C₆H₅Na₃O₇/C₆H₈O₇)    0-15 %Sodium perborate (as NaBO₃.H₂O)    11-18% TAED    2-6%Carboxymethylcellulose    0-2% Polymers (e g. maleic/acrylic acidcopolymer,    0-3% PVP, PEG) Enzymes (calculated as pure enzyme protein)0.0001-0.1% Minor ingredients (e.g. suds suppressors, perfume,    0-5%optical brightener, photobleach)

2) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

Linear alkylbenzenesulfonate (calculated as acid)    6-11% Alcoholethoxysulfate (e.g. C₁₂₋₁₈ alcohol, 1-2 EO    1-3% or alkyl sulfate(e.g. C₁₆₋₁₈₎ Alcohol ethoxylate (e.g. C₁₄₋₁₅ alcohol, 7 EO)    5-9%Sodium carbonate (as Na₂CO₃)    15-21% Soluble silicate (as Na₂O,2SiO₂)   1-4% Zeolite (as NaAlSiO₄)    24-34% Sodium sulfate (as Na₂SO₄)   4-10% Sodium citrate/citric acid (as C₆H₅Na₃O₇/C₆H₈O₇)    0-15%Carboxymethylcellulose    0-2% Polymers (e.g. maleic/acrylic acidcopolymer,    1-6% PVP, PEG) Enzymes (calculated as pure enzyme protein)0.0001-0.1% Minor ingredients (e.g. suds suppressors,    0-5 perfume)

3) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

Linear alkylbenzenesulfonate (calculated as acid)    5-9% Alcoholethoxylate (e.g. C₁₂₋₁₅ alcohol, 7 EO)    7-14% Soap as fatty acid (e.g.C₁₆₋₂₂ fatty acid)    1-3% Sodium carbonate (as Na₂CO₃)    10-17%Soluble silicate (as Na₂O,2SiO₂)    3-9% Zeolite (as NaAlSiO₄)    23-33%Sodium sulfate (as Na₂SO₄)    0-4% Sodium perborate (as NaBO₃.H₂O)   8-16% TAED    2-8% Phosphonate (e.g. EDTMPA)    0-1%Carboxymethylcellulose    0-2% Polymers (e.g. maleic/acrylic acidcopolymer,    0-3% PVP, PEG) Enzymes (calculated as pure enzyme protein)0.0001-0.1% Minor ingredients (e.g. suds. suppressors,    0-5% perfume,optical brightener)

4) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

Linear alkylbenzenesulfonate (calculated as acid)    8-12% Alcoholethoxylate (e.g. C₁₂₋₁₅ alcohol, 7 EO)    10-25% Sodium carbonate (asNa₂CO₃)    14-22% Soluble silicate (as Na₂O,2SiO₂)    1-5% Zeolite (asNaAlSiO₄)    25-35% Sodium sulfate (as Na₂SO₄)    0-10%Carboxymethylcellulose    0-2% Polymers (e.g. maleic/acrylic acidcopolymer,    1-3% PVP, PEG) Enzymes (calculated as pure enzyme protein)0.0001-0.1% Minor ingredients (e. g. suds suppressors,    0-5 perfume)

5) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

Fatty alcohol sulfate    5-10% Ethoxylated fatty acid monoethanolamide   3-9% Soap as fatty acid    0-3% Sodium carbonate (as Na₂CO₃)    5-10%Soluble silicate (as Na₂O,2SiO₂)    1-4% Zeolite (as NaAlSiO₄)    20-40%Sodium sulfate (as Na₂SO₄)    2-8% Sodium perborate (as NaBO₃.H₂O)   12-18% TAED    2-7% Polymers (e g. maleic/acrylic acid copolymer,   1-5% PEG) Enzymes (calculated as pure enzyme protein) 0.0001-0.1%Minor ingredients (e.g. optical brightener, suds    0-5% suppressors,perfume)

6) A detergent composition formulated as a granulate comprising

Linear alkylbenzenesulfonate (calculated as acid)    8-14% Ethoxylatedfatty acid monoethanolamide    5-11% Soap as fatty acid    0-3% Sodiumcarbonate (as Na₂CO₃)    4-10% Soluble silicate (as Na₂O,2SiO₂)    1-4%Zeolite (as NaAlSiO₄)    30-50% Sodium sulfate (as Na₂SO₄)    3-11%Sodium citrate (as C₆H₅Na₃O₇)    5-12% Polymers (e.g. PVP,maleic/acrylic acid    1-5% copolymer, PEG) Enzymes (calculated as pureenzyme protein) 0.0001-0.1% Minor ingredients (e. g. suds suppressors,   0-5% perfume)

7) A detergent composition formulated as a granulate comprising

Linear alkylbenzenesulfonate (calculated as acid)    6-12% Nonionicsurfactant    1-4% Soap as fatty acid    2-6% Sodium carbonate (asNa₂CO₃)    14-22% Zeolite (as NaAlSiO₄)    18-32% Sodium sulfate (asNa₂SO₄)    5-20% Sodium citrate (as C₆H₅Na₃O₇)    3-8% Sodium perborate(as NaBO₃.H₂O)    4-9% Bleach activator (e.g. NOBS or TAED)    1-5%Carboxymethylcellulose    0-2% Polymers (e.g. polycarboxylate or PEG)   1-5% Enzymes (calculated as pure enzyme protein) 0.0001-0.1% Minoringredients (e.g. optical brightener,    0-5% perfume)

8) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

Anionic surfactant (linear alkylbenzenesulfonate,    25-40% alkylsulfate, alpha-olefinsulfonate, alpha-sulfo fatty acid methyl esters,alkanesulfonates, soap) Nonionic surfactant (e.g. alcohol ethoxytate)   1-10% Sodium carbonate (as Na₂CO₃)    8-25% Soluble silicates (asNa₂O, 2SiO₂)    5-15% Sodium sulfate (as Na₂SO₄)    0-5 Zeolite (asNaAlSiO₄)    15-28% Sodium perborate (as NaBO₃.₄H₂O)    0-20% Bleachactivator (TAED or NOBS)    0-5% Enzymes (calculated as pure enzymeprotein) 0.0001-0.1% Minor ingredients (e.g. perfume, optical    0-3%brighteners)

9) Detergent formulations as described in 1)-8) wherein all or part ofthe linear alkylbenzenesulfonate is replaced by (C₁₂-C₁₈) alkyl sulfate.

10) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

(C₁₂₋C₁₈) alkyl sulfate    9-15% Alcohol ethoxylate    3-6% Polyhydroxyalkyl fatty acid amide    1-5% Zeolite (as NaAlSiO₄)    10-20% Layereddisilicate (e.g. SK56 from Hoechst)    10-20% Sodium carbonate (asNa₂CO₃)    3-12% Soluble silicate (as Na₂O,2SiO₂)    0-6% Sodium citrate   4-8% Sodium percarbonate    13-22% TAED    3-8% Polymers (e.g.polycarboxylates and PVP)    0-5% Enzymes (calculated as pure enzymeprotein) 0.0001-0.1% Minor ingredients (e.g. optical brightener, photo   0-5% bleach, perfume, suds suppressors)

11) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

(C₁₂-C₁₈) alkyl sulfate    4-8% Alcohol ethoxylate    11-15% Soap   1-4% Zeolite MAP or zeolite A    35-45% Sodium carbonate (as Na₂CO₃)   2-8% Soluble silicate (as Na₂O,2SiO₂)    0-4% Sodium percarbonate   13-22% TAED    1-8% Carboxymethyl cellulose    0-3% Polymers (e.g.polycarboxylates and PVP)    0-3% Enzymes (calculated as pure enzymeprotein) 0.0001-0.1% Minor ingredients (e. g. optical bnghtener,    0-3%phosphonate, perfume)

12) Detergent formulations as described in 1)-11) which contain astabilized or encapsulated peracid, either as an additional component oras a substitute for already specified bleach systems.

13) Detergent compositions as described in 1), 3), 5), 7) and 8) whereinperborate is replaced by percarbonate.

14) Detergent compositions as described in 1), 3), 5), 7), 8), 10) and11) which additionally contain a manganese catalyst. The manganesecatalyst may, e.g., be one of the compounds described in “Efficientmanganese catalysts for low-temperature bleaching”, Nature 369, 1994,pp. 637-639.

Particular forms of dishwashing detergent compositions within the scopeof the invention include:

1) Powder Automatic Dishwashing Composition

Nonionic surfactant   0.4-2.5% Sodium metasilicate    0-20% Sodiumdisilicate    3-20% Sodium triphosphate    20-40% Sodium carbonate   0-20% Sodium perborate    2-9% Tetraacetylethylenediamine (TAED)   1-4% Sodium sulphate    5-33% Enzymes 0.0001-0.1%

2) Powder Automatic Dishwashing Composition

Nonionic surfactant (e.g. alcohol ethoxylate)    1-2% Sodium disilicate   2-30% Sodium carbonate    10-50% Sodium phosphonate    0-5% Trisodiumcitrate dihydrate    9-30% Nitrilotrisodium acetate (NTA)    0-20%Sodium perborate monohydrate    5-10% Tetraacetylethylenediamine (TAED)   1-2% Polyacrylate polymer (e.g. maleic acidlacrylic acid    6-25%copolymer) Enzymes 0.0001-0.1% Perfume   0.1-0.5% Water    5-10

3) Powder Automatic Dishwashing Composition

Nonionic surfactant   0.5-2.0% Sodium disilicate    25-40% Sodiumcitrate    30-55% Sodium carbonate    0-29% Sodium bicarbonate    0-20%Sodium perborate monohydrate    0-15% Tetraacetylethylenediamine (TAED)   0-6% Maleic acid/acrylic acid copolymer    0-5% Clay    1-3%Poly(amino acids)    0-20% Sodium polyacrylate    0-8% Enzymes0.0001-0.1%

4) Powder Automatic Dishwashing Composition

Nonionic surfactant    1-2% Zeolite MAP    15-42% Sodium disilicate   30-34% Sodium citrate    0-12% Sodium carbonate    0-20% Sodiumperborate monohydrate    7-15% Tetraacetylethylenediamine (TAED)    0-3%Polymer    0-4% Maleic acid/acrylic acid copolymer    0-5% Organicphosphonate    0-4% Clay    1-2% Enzymes 0.0001-0.1% Sodium sulphateBalance

5) Powder Automatic Dishwashing Composition

Nonionic surfactant    1-7% Sodium disilicate    18-30% Trisodiumcitrate    10-24% Sodium carbonate    12-20% Monopersulphate (2KHSO₅.KHSO₄.K₂SO₄)    15-21% Bleach stabilizer   0.1-2% Maleicacid/acrylic acid copolymer    0-6% Diethylenetriaminepentaacetate,pentasodium salt    0-25% Enzymes 0.0001-0.1% Sodium sulphate, waterBalance

6) Automatic dishwashing compositions as described in 1), 2), 3) and 4),wherein perborate is replaced by percarbonate.

7) Automatic dishwashing compositions as described in 1)-4) whichadditionally contain a manganese catalyst. The manganese catalyst may,e.g., be one of the compounds described in “Efficient manganesecatalysts for low-temperature bleaching”, Nature 369, 1994, pp. 637-639.

All of the particular forms of washing and dishwashing detergentcompositions listed above will, of course, additionally comprise minoramounts of granulate constituents (core material, coating constituents,etc.)

The enzyme-containing granules of the invention may be incorporated inconcentrations corresponding to enzyme concentrations conventionallyemployed in detergents. It is at present contemplated that, in adetergent composition of the invention, the enzymes may be added in anamount corresponding to 0.00001-1 mg (calculated as pure enzyme protein)of enzyme per liter of wash/dishwash liquor.

Reference is made to WO 97/07202 (PCT/DK96/00341) for details concerningfurther types of detergent compositions relevant in the context of thepresent invention.

The present invention is further illustrated by the working examplesdescribed below, which are representative and are not intended to limitthe scope of the invention in any way. One skilled in the art will becapable of selecting other enzymes, cores, coating agents/adjuvants ormethods on the basis of the teaching herein.

MATERIALS AND METHODS

Enzyme activity assays

Proteolytic Activity KNPU): In the present specification, proteolyticactivity is expressed in Kilo Novo Protease Units (KNPU). The activityis determined relative to an enzyme standard, and the determination isbased on the digestion of a dimethyl-casein (DMC) solution by theproteolytic enzyme under standard conditions (50° C., pH 8.3, 9 min.reaction time, 3 min. measurement time). A brochure (AF 220/1) providingfurther details of the analytical method is available upon request fromNovo Nordisk A/S, Bagsvaerd, Denmark.

Amylolytic activity (KNU) may be determined using potato starch assubstrate. This method is based on the hydrolysis of modified potatostarch by the enzyme, and the reaction is followed by mixing samples ofthe starch/enzyme solution with an iodine solution. Initially, ablackish-blue colour is formed, but during the breakdown of the starchthe blue colour becomes weaker and gradually changes to a reddish-brown,which is compared to a coloured glass standard. One Kilo Novo alphaAmylase Unit (KNU) is defined as the amount of enzyme which, understandard conditions (37±0.05° C., 0.0003 M Ca²+, pH 5.6) dextrinizes5.26 g starch dry substance Merck Amylum solubile.

A brochure (AF 9/6) describing this analytical method in more detail isavailable upon request from Novo Nordisk A/S, Denmark.

Lipolytic activity (LU) may be determined using tributyrin as substrate.This method is based on the hydrolysis of tributyrin catalyzed by theenzyme, and the consumption of alkali is measured as a function of time.One Lipase Unit (LU) is defined as the amount of enzyme which, understandard conditions (30.0° C., pH 7.0; Gum Arabic as emulsifier andtributyrin as substrate) liberates 1 mmol of titratable butyric acid perminute. A brochure (AF 95/5) describing this analytical method in moredetail is available upon request from Novo Nordisk A/S, Denmark.

Phytase activity (FYT) may be determined using Novo Nordisk analyticalmethod KAL-SM-0403.01/01 (available upon request from Novo Nordisk A/S,Denmark).

Cores

Cassava starch cores: If nothing else is stated, cassava starch coresemployed were obtained from Agro Comercial, Brazil.

Nonpareil cores (sugar-starch based): obtained from Crompton & Knowles,USA (“Sugar Spheres NF Mesh 35-40”) or from NP Pharma.

Enzymes

Savinase™ concentrate (water-based protease concentrate) was obtainedfrom Novo Nordisk A/S, Denmark.

Phytase concentrate was obtained from Novo Nordisk A/S, Denmark. Lipase(lipase variant HL9) was obtained as described in Example 3 of WO97/07202.

Heubach method and Novo Nordisk attrition method

As already mentioned (vide supra), both the Heubach method and the NovoNordisk attrition method are methods wherein a bed of granules isexposed to the action of rolling steel balls with simultaneous suctionof air through the bed to collect dust and fragments created during theprocess. Brochures (EAL-SM-0289.01/01 and AF 225/2-GB, respectively)describing these methods in more detail are available upon request fromNovo Nordisk A/S, Denmark.

EXAMPLE 1

A 15 kg portion of granular cassava starch cores (particle sizedistribution: 97% of the material between 0.5 mm and 1.0 mm in diameter)was introduced into a 50 liter Lodige mixer and sprayed, with mixing bymeans of the mixing blades (“ploughshares”), with a total of 6.0 kg ofpure water (40% w/w relative to the cores), initially by spraying on 3.5kg of water without operating the “chopper” (compacting knives) and thenby spraying on the remaining amount of water (2.5 kg), while operatingthe chopper, up the total amount of 6.0 kg.

The wetted cores were inspected regularly during the spraying and showedno sign of stickiness or tendency towards agglomeration which couldcause problems during subsequent drying or problems with respect to thequality of the final product.

The resulting product was transferred to a Glatt WSG 15 fluid-bedapparatus (Glatt, Germany) with an air-inlet temperature of 62° C., anddried for 30 minutes, or until the product temperature exceeded 50° C.,and then sieved on a 1.2 mm mesh screen, leaving only 0.8% w/w ofresidual, oversized particles on the sieve.

EXAMPLE 2

(comparative example using a poorly absorbing core)

A 15 kg portion of sugar-starch nonpareil cores was introduced into a 50liter Lodige mixer and sprayed, with mixing by means of the mixingblades, stepwise with a total of 0.9 kg of water, starting with 0.3 kg.

The wetted cores were inspected regularly during the spraying. Afterspraying of the first 0.3 kg of water (2% w/w relative to the cores) thecores showed stickiness. By the time a total of 4-5% w/w of water hadbeen sprayed on, stickiness was severe.

After spraying a total of 0.9 kg of water (6% w/w relative to the cores)the wetted cores were transferred to a Glatt WSG 15 fluid-bed apparatusfor drying under the same conditions as in Example 1. However, even whenattempts were made to mix the bed of wetted cores manually in thefluid-bed apparatus, the material proved to be too sticky to fluidizesatisfactorily. The final dried product consisted mainly of agglomeratedlumps or crusts with dimensions of up to about 10 cm.

This example demonstrates that the nonpareil cores in question arecapable of absorbing less than 4% w/w of water.

EXAMPLE 3

Step 1: A 20 kg portion of granular cassava starch cores (as employed inExample 1; particle size distribution: 97% of the material between 0.5and 1.0 mm in diameter) was introduced into a 50 liter Lödige mixerequipped with a multiple chopper head, where they were sprayed, withmixing by means of the mixing blades, with 4.5 kg of water-based,ultrafiltered liquid Savinase:™ concentrate (33 KNPU/g). The Savinase™concentrate was applied using a pressure nozzle submerged in the coresand spraying directly into the chopper. The mixing blades and thechopper were operated continuously during the spraying.

After applying the Savinase™ concentrate, the resulting granules weretransferred to a Glatt WSG 5 fluid-bed apparatus and dried as describedin Example 1.

Step 2: A 15 kg portion of the dried granulate was subsequently coatedin the fluid-bed apparatus with 8% w/w of ammonium sulphate, using a 38%w/w ammonium sulphate solution and employing a conventional top spraycoating technique (air inlet temperature 62° C., product temperature 43°C., spray rate 100 g/min, air flow 700 m³/h).

Step 3: The ammonium sulphate coated granulate was further coated with18.5% w/w of a coating of composition 27% TiO₂, 27% kaolin, 30% Glascol™LS27 and 16% PEG 4000, using the same fluid bed top spray coatingtechnique. The composition of the coating liquid was:

0.75 kg TiO₂ (Kronos™ 2044)

0.75 kg kaolin (Speswhite™ from English China Clay)

1.80 kg Glascol™ LS27 (from Allied Colloids Ltd., England; 46% drymatter)

0.45 kg Polyethylene glycol 4000 (PEG 4000)

2.40 kg water.

Step 4: Finally a surface coating of 0.75% w/w of PEG 4000 was appliedusing a 23% w/w water-based solution of PEG 4000 and employing the samefluid bed top spray technique as before.

The activity of the final granulate product after step 4 was 4.1 KNPU/g.

The dust-formation/physical strength properties of the granulate afterstep 1 and step 2 were evaluated according to the Novo Nordisk attritionmethod, whilst the products resulting from step 3 and step 4 wereevaluated according to the Heubach method.

Dust content after:

Step 1: Total dust 0.4 mg; active dust 2410 μg

Step 2: Total dust 0.5 mg; active dust 74 μg

Step 3: Total dust 6.2 mg; active dust 43 μg

Step 4: Total dust 0.0 mg; active dust <10 μg

The amount of active dust is expressed as micrograms (μg) of a standardwith an activity of 4.0 KNPU/g.

EXAMPLE 4

Step 1: A 15 kg portion of granular cassava starch cores (as employed inExample 1 and Example 3) was introduced into a 50 liter Lodige mixer,and sprayed, with mixing by means of the mixing blades, with 5.9 kg ofliquid Savinase™ concentrate (33 KNPU/g) and dried as described inExample 1.

Steps 2-4: A 15 kg portion of the dried granulate from step 1 wastreated as described in steps 2-4 in Example 3.

The activity of the final granulate product after step 4 was 10.0 KNPU/g

The dust-formation properties of the products of steps 1-4 (determinedas in Example 3) were as follows:

Step 1: Total dust 5.9 mg; active dust 7330 μg

Step 2: Total dust 0.0 mg; active dust 78 μg

Step 3: Total dust 2.6 mg; active dust <10 μg

Step 4: Total dust 0.0 mg; active dust <10 μg

EXAMPLE 5

A 15 kg portion of granular cassava starch cores (as employed inExample 1) was introduced into a 50 liter Lodige mixer and sprayed, withmixing by means of the mixing blades, with 5.9 kg of liquid Savinase™concentrate (18 KNPU/g) using a pneumatic nozzle placed in the funnelabove the mixer. In this example the chopper was not operated during thespraying, but the sprayed granulate was subjected to treatment with thechopper for 3 minutes after the spraying.

The activity of the resulting granulate (uncoated granulate) was 6.28KNPU/g, and the dust-formation properties of the product (determined bythe. Novo Nordisk attrition method) were as follows:

Total dust 0.5 mg; active dust 483 4μg.

EXAMPLE 6

(comparative example using poorly absorbing cores)

In experiments using nonpareil cores (sugar-starch cores) sprayedsuccessively with increasing amounts of liquid Savinase™ concentrate inthe same manner as described in Examples 3, 4 and 5, the presence ofsurplus concentrate on the outer surface of the cores, with attendantparticle agglomeration, was observed when ≧4% w/w (relative to thecores) of concentrate was applied.

This demonstrates that the nonpareil cores in question are able toabsorb less than 4% w/w of the Savinase™ concentrate.

EXAMPLE 7

This example is a comparative example in which poorly absorbing,nonpareil (sugar-starch) cores are sprayed with an aqueous,enzyme-containing medium, and dried, in a conventional fluid-bedapparatus.

Step 1: A 15 kg portion of nonpareil cores (particle size distribution:99% between 425 μm and 600 μm in diameter) was introduced into a GlattWSG 5 fluid-bed apparatus and sprayed, under fluidized conditions, witha mixture of 4.0 kg of liquid Savinase™ concentrate (33 KNPU/g), 225 gof 20% Kollidon™ VA64 and 50 g of TiO₂ using a conventional top spraycoating technique (inlet air temperature 65° C.; product temperature 42°C.;, spraying rate 100 g/minute; air flow 550 m³/hour).

The product was then fluid-bed dried as described above.

Steps 2-4: The product from step 1 was treated successively as in steps2-4 in Example 3.

The activity of the final product was 6.2 KNPU/g.

The dust-formation properties of the various products (determined usingthe Novo Nordisk attrition method for the products from step 1 and step2, and the Heubach method for the products from step 3 and step 4) wereas follows:

Step 1: Total dust 9.1 mg; active dust 56900 μg

Step 2: Total dust 23.2 mg; active dust 72400 μg

Step 3: Total dust 12.3 mg; active dust 6660 μg

Step 4: Total dust 1.8 mg; active dust 1570 μg

It is apparent from these results that irrespective of the level ofcoating, the various products—based on poorly absorbent nonpareilcores—exhibit unfavourable dust-formation properties.

EXAMPLE 8

18 kg of a powder composition prepared from the following:

4.5 kg of fibrous cellulose (Arbocel™ FTC200),

3.0 kg of kaolin (Speswhite™, English China Clay) and

20.5 kg of finely ground sodium sulphate were sprayed with 9.5 kg of a21% w/w aqueous solution of carbohydrate binder (Glucidex™ 21D, fromRoquette Freres) and granulated and dried as described in Example 1 inU.S. Pat. No. 4,106,991.

The dry granulate was sieved, and the size fraction between 0.3 and 1.0nun was used as enzyme-absorbing core in the following:

Step 1: A 18.5 kg portion of the core material was transferred to a 50liter Lödige mixer and sprayed, with mixing by means of the mixingblades, with 4.5 kg of liquid Savinase™ concentrate. The product wasdried as described in Example 3.

Steps 2-4: 16 kg of granulate from step 1 was transferred to a GlattWSG5 fluid bed apparatus, and then dried as described above and treatedfurther as described in steps 2-4 in Example 3.

The activity of the final product was 5.5 KNPU/g.

Dust-formation properties (determined using the Novo Nordisk attritionmethod for the products from step 1 and step 2, and the Heubach methodfor the products from step 3 and step 4) were as follows:

Step 1: Total dust 11.6 mg; active dust 29900 μg

Step 2: Total dust 0.7 mg; active dust 741 μg

Step 3: Total dust 7.3 mg; active dust 367 μg

Step 4: Total dust 0.6 mg; active dust 51 μg

EXAMPLE 9

Step 1: A 15 kg portion of granular cassava starch cores (as employed inExample 1) was introduced into a 50 liter Lodige mixer equipped with amultiple chopper head, where they were sprayed, with mixing by means ofthe mixing blades, with 5.0 kg of ultrafiltered Savinase concentrate(27.4 KNPU/g). The concentrate was sprayed into the cores as describedin Example 3.

After applying the. Savinase™ concentrate, the resulting granularmaterial was transferred to a Glatt WSG 15 fluid-bed apparatus and driedas described above.

Step 2: After drying, the resulting granulate was coated with a thinfilm coating (2% w/w in total) using a Wurster-type fluid- bedapparatus: 15 kg of the granulate from step 1 was transferred to a GlattGRPC 15 fluid-bed apparatus (bottom spray) where it was sprayed with amixture of 150 g of methylhydroxypropylcellulose (Aqualon™ 8MP5C) and150 g of PEG 4000 dissolved in 1.8 liters of water. Air inlettemperature: 55° C.; product temperature: 39.5° C. After spraying, thegranulate was dried for 5 minutes and then cooled to 30° C.

The activity after the final step 2 was 7.0 KNPU/g.

Dust content after:

Step 1: Total dust 0.2 mg; Active dust 263 μg (Novo Nordisk attritionmethod)

Step 2: Total dust 0.0 mg; Active dust 26 μg (Heubach method)

These very low dust figures clearly demonstrate the physical quality ofthe enzyme/cassava core granulate, since only a thin film coating hadbeen applied.

EXAMPLE 10

Step 1: A 15 kg portion of cassava starch cores (as employed inExample 1) was introduced into a Glatt WSG 5 fluid-bed apparatus andsprayed with a mixture consisting of 3.9 kg of liquid Savinase™concentrate (27.4 KNPU/g), 200 g of a 20% w/w solution of Kollidon™ VA64in water, and 50 g of TiO₂, as described in Example 7, step 1. Step 2-4:The product was treated (coated) as described in Examples 3 and 7, steps2-4.

The activity of the final product was 5.9 KNPU/g.

The dust-formation properties of the products of steps 1-4 (determinedas in Example 3) were as follows:

Step 1: Total dust 0.0 mg; active dust 474 μg

Step 2: Total dust 1.7 mg; active dust 1550 μg

Step 3: Total dust 3.3 mg; active dust 294 μg

Step 4: Total dust 0.0 mg; active dust <10 μg

This example demonstrates that the contacting/absorption of step (a) ofthe process according to the invention may be performed in a fluid-bedand still yield a product exhibiting excellent properties (such as lowdust formation). In contrast, the similar procedure described in Example7 (vide supra), using a poorly absorbing non-pareil core, gave productsexhibiting relatively high dust levels.

In addition to absorption properties, a combination of one or more offactors such as high physical strength, high degree of sphericity,smoothness and degree of starch gelatinization of the absorbent cores inquestion is believed to contribute to the low dust figures observed forproducts (products according to the invention) exemplified by thoseobtained in the present example, and in other examples herein (e.g.Example 3), which employ cores of a type preferred in the context of theinvention.

EXAMPLE 11

In this example a liquid enzyme was partly absorbed into, partly layeredonto, a cassava core, the entire process being carried out in aHuttlin-type fluidizer with bottom spray.

Step 1: A 3.5 kg portion of cassava starch cores (as employed inExample 1) was introduced into a 5 liter Huttlin Turbojet fluidizer typeHKC-5-TJ, where they were sprayed with 1500 g of liquid Savinase™concentrate (27.4 KNPU/g), keeping the product temperature at 24° C. byslowly raising the inlet temperature from 35° C. to 65° C. At this pointthe granulate was too wet to fluidize properly, and it was consequentlydried by stopping the spraying and allowing the product temperature torise to 40° C. After drying, the product was sprayed with a further 1600g of the Savinase™ concentrate (inlet air temperature 80° C.; producttemperature 44° C.). After the spraying, the product was dried bycontinuing the passage of inlet air (80° C.) for 2 minutes, and thencooled.

Step 2: The dried granulate from step 1 was subsequently coated in thefluid-bed apparatus with 8% w/w of ammonium sulphate by spraying with a38% w/w aqueous ammonium sulphate solution (inlet air temperature 78°C.; product temperature 38° C.).

Step 3: The ammonium sulphate coated granulate was further coated with18.5% w/w of a coating with a composition as described in Example 3,step 3, the composition of the coating liquid employed being the same asemployed in step 3 of Example 3.

Step 4: Finally a surface coating was applied as described in Example 3step 4.

The activity of the final granulate after step 4 was 18.5 KNPU/g.

Dust-formation properties of the final product (Heubach method):

Total dust: 0.4 mg; active dust: 28 μg

The results demonstrate that the product (a product according to theinvention which not only has enzyme absorbed within the core, but alsohas enzyme deposited on the outer surface thereof) obtained by thisprocedure (a process within the scope of the present invention) not onlyhas a very high enzyme content (and thereby very high activity), butalso exhibits very low tendency towards dust formation.

Without being bound to any theory, it is believed that the adherence ofenzyme deposited on the outer surface of an absorbent core of the typeof relevance in the context of the invention is enhanced by thepresence, within the surface of the core, of absorbed enzyme, and thusthat the tendency to dust formation by such a product is correspondinglyreduced.

EXAMPLE 12

Step2 1: A 15 kg portion of cassava starch cores (as employed inExample 1) was introduced into a 50 liter Lodige mixer equipped with amultiple chopper head, and the cores were sprayed, with mixing by meansof the mixing blades, with a total of 5.0 kg of an ultrafiltered lipaseconcentrate with an activity of 145 KLU/g. The preparation of the lipasein question is described in WO 97107202 (see Example 3, variant HL9,therein). The concentrate was sprayed from a two-fluid nozzle (airatomizer nozzle) placed in the funnel above the mixer.

After the lipase concentrate had been applied, the resulting granularmaterial was transferred to a Glatt WSG 5 fluid-bed apparatus and driedas described previously, above.

Step 2: After drying, the resulting granulate was coated with 4.8% w/wof PEG 4000 and 12.5% w/w of a 1:1 mixture of titanium dioxide andkaolin, using a procedure as described in U.S. Pat. No. 4,106,991(Example XXII therein), with the exception that PEG 4000 was used hereininstead of PEG 1500.

The activity of the final granulate product after step 2 was 32 KLU/g.

EXAMPLE 13

This example describes the preparation of an enzyme-containing granulateof a known type (viz. a granulate in accordance with U.S. Pat. No.4,106,991) for comparison purposes (see Example 14, below).

Step 1: A powder mixture with the following composition:

2.25 kg of fibrous cellulose (Arbocel™ BFC200)

1.50 kg of kaolin (Speswhite™, from English China Clay)

1.00 kg of carbohydrate binder (Glucidex™ 21D, Roquette Freres)

9.35 kg of ground sodium sulphate

was sprayed with 3.0 kg of Lipase concentrate (as employed in Example12) in which was further dissolved 0.4 kg of carbohydrate binder(Glucidex™ 21D). The mixture was granulated and dried as described inU.S. Pat. No. 4,106,991 (Example I therein).

Step 2: The dry granulate was sieved to obtain a product with a particlesize range of 0.3-1.2 mm; this product was subsequently coated asdescribed in Example 12, step 2.

The activity of the final granulate product after step 2 was 20 KLU/g.

EXAMPLE 14

This example compares the storage stability in detergents of (i) alipase-containing, starch-based granulate in accordance with the presentinvention (Example 12, above), and (ii) a lipase-containing granulateproduced in accordance with U.S. Pat. No. 4,106,991 (Example 13, above).

A: Storage in a perborate-containing detergent with TAED

Products produced according to Example 12 and Example 13, respectively,were mixed (to an enzyme content of ca. 1 KLU/g of detergent) into atraditional (non-compact), zeolite-built powder detergent containingsodium perborate and tetraacetylethylenediamine (TAED). Analyticalresults (residual lipase activity) obtained after storage of theresulting enzyme-containing detergent composition under variousconditions are given in the tables below:

(i) Storage at 35 ° C. and 55% relative humidity in open jars

% residual activity (2 determinations) after Granulate 2 weeks 4 weeksExample 12 93-96 101-105 Example 13 80-84 78-71

(ii) Storage at 37° C. and 70% relative humidity in open jars

% residual activity after Granulate 3 days 7 days 14 days Example 12 10090 91-87 Example 13 84 68 49-43

B: Storage in a percarbonate-containing detergent

Products produced according to Example 12 and Example 13, respectively,were mixed (to an enzyme content of ca. 1 KLU/g of detergent) into astandard, compact, European-type powder detergent containing sodiumpercarbonate. Analytical results (residual lipase activity) obtainedafter storage of the resulting enzyme-containing detergent compositionunder specified conditions are given in the table below:

(i) Storage at 35° C. and 55% relative humidity in open jars

% residual activity after Granulate 2 weeks 4 weeks Example 12  97-10092-89 Example 13 58-52 36-35

EXAMPLE 15

This example describes the preparation of absorbent cores (enzyme-free)of a type. similar to those produced in Example 8, but containing ricestarch instead of sodium sulphate.

A 14 kg portion of ground, recycled, enzyme-free cores (produced usingthe methodology of EP 0 304 331 B1, but without incorporating enzyme)with a composition corresponding to that of a mixture of the followingamounts of the following powder components was mixed with the powdercomponents in question, viz.

3.15 kg of fibrous cellulose (Arbocel™ BFC200)

2.10 kg of kaolin (Speswhite™, from English China Clay) and

20 12.75 kg of rice starch (from Remy Industri).

The dry mixture was sprayed with 14 kg of a 21.4% w/w solution ofcarbohydrate binder (Glucidex 21D) in water, and granulated and dried asdescribed in EP 0 304 331 B1 (Example 1 therein).

The dry granulate (cores) was sieved to obtain cores with a particlesize in the range of 0.3-1 mm.

EXAMPLE 16

Step 1: A 15 kg portion of the sieved cores from Example 15 was sprayedwith 3.3 kg of Savinase™ concentrate (33 KNPU/g) in a Lodige mixer asdescribed in Example 3. The thus wetted granulate was then powdered, inthe mixer, with 300 g of kaolin (Speswhite™). The product was thentransferred to a Marumerizer™ (from Fuji Paudal, Osaka, Japan), where itwas further spheronized. The resulting granulate was dried in a GlattWSG 5 fluid-bed apparatus essentially as in Example 1.

Steps 2-4: The dried granulate was coated as described in Example 3,steps 2-4.

The activity of the final granulate after step 4 was 4.7 KNPU/g.

Dust content after:

Step 1: Total dust 0.4 mg; active dust 1000 μg (Novo Nordisk attritionmethod).

Step. 2: Total dust 1.0 mg; active dust 194 μg (Novo Nordisk attritionmethod).

Step 4: Total dust 0.6 mg; active dust <10 μg (Heubach method).

EXAMPLE 17

Step 1: A 15 kg portion of cassava starch cores (as employed inExample 1) in a 50 liter Lödige mixer was sprayed, with mixing by meansof the mixing blades, with 5.0 kg of Savinase™ concentrate (33 KNPU/g)and dried as described in Example 3, with the exception that the spraynozzle did not spray directly into the chopper but from the funnel abovethe mixer.

Step 2: A 2.0 kg portion of dried granulate was transferred to a 5 literLödige mixer and coated with 3% w/w of PEG 4000 and 3% w/w of a 1:1mixture of titanium dioxide and kaolin in a manner as described in U.S.Pat. No. 4,106,991 (Example XXII therein).

The final granulate had an activity of 10.8 KNPU/g and a dust content(according to the Heubach method) of 0.2 mg total dust and 37 g activedust.

EXAMPLE 18

A granulate was prepared as described in Example 17, above, except thatthe final coating consisted of 5% w/w of PEG 4000 and 13% w/w of a 1:1mixture of titanium dioxide and kaolin.

The product granulate had an activity of 10.5 KNPU/g, and had a dustcontent (Heubach) of 0.4 mg total dust and <10 μg active dust.

By way of comparison, it may be mentioned that a granulate producedaccording to U.S. Pat. No. 4,106,991 and with a coating of the typedescribed in the present example generally exhibits dust figures ofaround 200-300 μg active dust.

EXAMPLE 19

Enzyme-free cores (absorbent cores) of composition:

10.0% w/w of fibrous cellulose (Arbocel BC200)

10.0% w/w of kaolin (Speswhite)

10.0% w/w of carbohydrate binder (3:2 mixture of Glucidex™ 21D andSorbitol) balance: sodium sulphate

was produced, by a continuous procedure, according to U.S. Pat. No.4,106,991, leading to cores which were not fully compacted and fullyspheronized. These cores were compacted and physically improved in thefollowing manner:

Step 1: An 18 kg portion of the (enzyme-free) cores was sprayed with 3.0kg of Savinase™ concentrate (27.4 KNPU/g), in which was dissolved 2% w/wof sodium thiosulphate, in a manner as described in Example 12, above.

After mixing the product for 2 minutes (operating both the mixer bladesand the chopper), the granulate was powdered with 600 g of rice starch(Remy Industri), and then sprayed with a further 500 g of Savinase™concentrate.

A further 600 g of rice starch was applied, followed by a furtherspraying with 500 g of Savinase™ concentrate.

Finally, the granulate was powdered with a further 600 g of rice starch,and then with 360 g of kaolin.

During all the latter procedures, the mixer blades and the chopper wereoperated in order to compact the granulate and smoothen the surface ofthe granules.

The granulate was then dried as described in Example 1.

Steps 2-3: A 15 kg portion of the dried granulate was coated asdescribed in Example 3, steps 2-3.

The activity of the final product after step 3 was 6.4 KNPU/g.

Dust content (Heubach): total dust 0,1 mg; active dust 32 μg.

This example demonstrates that the tendency to dust formation byenzyme-containing granules (granules according to the invention) basedon “placebo” cores (enzyme-free cores) prepared according to themethodology of U.S. Pat. No. 4,106,991 can be still further reduced bystarch coating followed by application/absorption of enzyme.

EXAMPLE 20

Sphericity of Cassava Starch Cores

This example gives the results of measurements (by microscopy) of thesphericity, expressed as the ratio between the largest diameter (dmax)and the smallest diameter (dmin), for each of 20 cassava starch corestaken at random from a batch of cores (size distribution: 97% between0.5 mm and 1.0 mm) supplied by μAgro Comercial, Brazil.

The results were as follows:

Particle No. d_(max)/d_(min) 1 1.056 2 1.029 3 1.086 4 1.025 5 1.188 61.852 7 1.117 8 1.032 9 1.025 10 1.056 11 1.056 12 1.081 13 1.104 141.244 15 1.091 16 1.027 17 1.031 18 1.030 19 1.063 20 1.056 Mean ofd_(max)/d_(min): 1.112

If particle No. 6, which deviates markedly from the others, is ignored,the mean sphericity of the remaining 19 particles is 1.074.

This example illustrates that absorbent cores which are among preferredtype of cores in the context of the invention, viz. cassava starchcores, are available in a highly spherical quality. Inspection of thesecores by microscopy also revealed a high degree of surface smoothness.

EXAMPLE 21

Determination of Degree of Gelatinization

The degree of gelatinization was estimated by measurements of thereduction in gelatinization enthalpy by differential scanningcalorimetry (DSC).

About 200 mg of (a) native cassave starch (starch grains) and (b)powdered (crushed) starch cores, respectively, were weighed intorespective DSC pans together with deionized water to give a 25% w/w (drysubstance) slurry. The samples were sealed and heated at 1° C./min from45° C. to 95° C. Air served as reference. Measurements were made using adifferential scanning calorimeter from Hart Scientific.

The degree of gelatinization was calculated according to A. Xu and P.A.Seib [Cereal Chem. 70(4) (1993) pp. 463-70] as:H_(cord)/ΔH_(native starch), where ΔH_(core) is the endothermic enthalpychange for the (powdered) cores, and ΔH_(native starch) is theendothermic enthalpy change for the native starch.

The table below gives results for different batches of cassava starchcores (substantially spherical cores) from various sources, togetherwith the estimated water-absorption capacity for the various cores(estimated, e.g., as in Example 1 herein):

% Gelatin- Absorption Core supplier ization capacity (% w/w) Cia. Lorenz(Brazil) 43 ≧33 Agro Comercial (Brazil) (1) 53 ≧33 Agro Comercial(Brazil) (2) 42 ≧33 Sukhjit (India) 92 <20

EXAMPLE 22

Trial production-scale Preparation of Savinase™/-cassava Starch Cores

327 kg of cassava starch cores (from Cia. Lorenz, Brazil) were chargedinto a 1200 liter Lödige mixer equipped with 5 choppers; however, thechoppers were not operated in the following:

A total of 81 kg of Savinase™ concentrate (activity 37.15 KNPU/g;dry-matter content 28.4% w/w) was sprayed onto the cores, with mixing bymeans of the mixing blades, at a feed rate of approximately 10 kg/min,using 2 nozzles placed in the “chimneys” in the upper part of the mixer.Mixing was continued for a further 5 minutes after stopping thespraying.

The product, which showed no sign of stickiness or tendency toagglomerate, was transferred to a drying fluid bed. After 10 minutes ofdrying employing air with a velocity of ca. 1.5 m/sec and an initial airinlet temperature of 60° C., the inlet air temperature was increased to75° C. for a further 10 minutes and then to 95° C. The total drying timewas 32 minutes, and the fluid bed was emptied when the bed/producttemperature reached 80° C.

The dried product (raw granulate) was then sieved on a three-deck sieve,and the fraction of size ca. 300-1100 μm was coated in a 600 literLödige mixer as follows:

318 kg of raw granulate was introduced into the mixer. 3.8% w/w(relative to the raw granulate) of PEG 4000 was added with mixing, andthe mixer was kept running for 1 minute. 12.5% w/w (relative to the rawgranulate) of a powder mix consisting of 38:5% w/w of titanium dioxideand 61.5% w/w of kaolin was then added. After operating the mixer for afurther 30 seconds, 0.5% w/w of PEG 4000 was added and the mixer waskept running for a further 1 minute before adding 3% w/w (relative tothe raw granulate) of the above-mentioned TiO₂/kaolin powder mix. Themixer was then operated for a further 2 minutes.

The resulting coated product was transferred to a cooling fluid bed (airvelocity ca. 1.5 m/s) with an air inlet temperature ranging from 150° C.to 200° C., and cooled therein for 31 minutes. The final producttemperature was 260° C.

The cooled product was then sieved on a two-deck sieve and finallybagged. A sample of the product was examined with respect to protease(Savinase™ activity and dust level, giving the following results:

Activity: 6.56 KNPU/g

Dust (Heubach): Total dust: 0.1 mg; active dust:43 μg

EXAMPLE 23

Performance of a Savinase™/Cassava Starch Granulate in Textile (laundry)Washing

In this example, the washing performance of a coated, Savinase™/cassavastarch granulate of the invention (prepared and coated essentially as inExample 3 (vide supra), but containing a higher level of Savinase™), wascompared with that of a standard, commercially available Savinase™granulate (coated granulate) of a type according to U.S. Pat. No.4,106,991 (Savinase™ 6.0T, from Novo Nordisk A/S, Denmark).

The Savinase™/cassava granulate (activity 6.81 KNPU/g) and the theSavinase™ 6.0T granulate (activity ca. 6.5 KNPU/g), respectively, weretested in conjunction with a detergent composition [Red OMO™ (compactpowder) from China] for performance with respect to stain-removal fromstandard test swatches (EMPA117, from Center for Test Materials,Holland; white cotton/polyester stained with blood, milk and carbonblack) under conditions corresponding to Japanese washing conditions(vide infra).

Red OMO™ as supplied contains enzymes, and for the purposes of the testsdescribed herein the enzyme content was deactivated before use, asfollows (amounts given are per single wash):

35 g of Red OMO™ compact powder as supplied was dissolved/-dispersed in400 ml of deionized water with stirring at ambient temperature for 10minutes. The solution/dispersion was then heated at 850° C. for 5minutes in a microwave oven before use in the washing procedure.

Each swatch was attached to a black T-shirt (100% cotton) which was thuswashed together with the swatch in order to examine whether any solidresidue (notably cassava starch) could be detected thereon afterwashing. In each wash, 9 swatches/T-shirts were washed together. Forcontrol purposes, a corresponding wash was performed without any enzyme(enzyme-containing granulate) added to the washing medium.

The washing conditions are summarized below: Detergent: Red OMO ™(enzyme content deactivated prior to use) Detergent dosage: 1.0 g/l pH:10.2-10.3 (not adjusted) Washing time: 12 minutes Washing temperature:20° C. Water hardness: 6° dH Ca²⁺/Mg²⁺ (2:1) Wash liquor volume: 35liters Enzyme concentration 10 nM in washing medium: Washing machine:Japanese Test fabric: EMPA117 swatches + black T-shirts (9swatches/T-shirts per wash)

Water of the correct hardness was prepared by adding calcium andmagnesium chloride to deionized water.

The reflectance/emission, R, of the test swatches was measured at 460 nmusing an Elrepho 2000 photometer (aperture 10 mm, without UV).

The results are summarized in the following table, which gives the meanof the R values for the 9 swatches/T-shirts in each wash:

Tested granulate Mean R value Savinase ™ /cassava starch 60.8 Savinase ™6.0 T 62.0 None (no enzyme) 47.5

In no case was any residue visible on the black T-shirt material.

The results indicate that the washing performance of the coatedSavinase™/cassava starch granulate according to the invention compareswell with that of the commercial Savinase™ 6.0T granulate.

EXAMPLE 24

Crushing Strength of Cores

In this example, the resistance of various types/fractions of cassavastarch cores (from Brazilian suppliers) and non-pareil cores to crushingwas determined using the apparatus shown in FIG. 1 (vide infra) in themanner described earlier, above.

The crushing strengths (in g/mm²) given in the table below are the meanof the values for 20 particles (cores) taken at random from a batch ofthe core type/fraction in question. Standard deviations (SD) are alsogiven.

Core type Size range Strength SD (supplier) (μm) (g/mm²) (g/mm²) Cassavastarch 500-600 1010 770 (Agro Comercial) Cassava starch 500-600 1495 619(Cia. Lorenz) Cassava starch 600-710 1038 816 (Agro Comercial) Cassavastarch 600-710 1662 754 (Cia. Lorenz) Cassava starch 710-850 1104 657(Agro Comercial) Cassava starch 710-850 1600 565 (Cia. Lorenz)Non-pareil 500-600 93 88 (Crompton & Knowles) Non-pareil 500-600 203 96(NP Pharma)

EXAMPLE 25

This example describes the preparation of coated, Savinase™-containinggranulates (granulates according to the invention) based on potatostarch cores and corn (maize) starch cores, respectively.

A: Preparation from potato starch cores:

Step 1: A 15 kg portion of potato starch cores with good absorptionproperties (from TIPIAK, France) with a particle size range of 1.8-3.2mm was introduced into a 50 liter Lödige mixer, where they were sprayed,with mixing at 150 rpm and without operating the chopper, with 5.0 kg ofultrafiltered Savinase™ concentrate (27.4 KNPU/g), using a two-fluidnozzle.

The product was then transferred to a Glatt WSG 5 fluid bed apparatusand dried as described previously.

Step 2: The dried granulate was coated with 4.8% w/w of PEG 4000 and12.5% w/w of a 1:1 mixture of titanium dioxide and kaolin, as describedin Example 12 (vide supra).

The active dust content (Heubach method) after coating was 35 μg.

B: Preparation from corn starch cores:

A 15 kg portion of corn starch cores (from Santos, India) with aparticle size range of 850-2000 m was treated with Savinase™concentrate, and subsequently dried, as described above in step 1 forpotato starch cores.

EXAMPLE 26

This example describes the preparation of coated phytase/cassava starchgranulates.

Granulate 1.

Step 1: A 3.5 kg portion of cassava starch cores (from Agro Comercial,Brazil; fraction of predominant size range 300-1000 μm) was introducedinto a 20 liter Lödige mixer. The cores were sprayed, with mixing at thehighest mixing speed of the apparatus, with a phytase solution preparedby dilution of a phytase concentrate (from Novo Nordisk A/S) to aconcentration of 10700 FYT/g. The chopper was not operated.

Step 2: The product from step 1 was transferred to a fluid bed and driedat 60° C.

Step 3: The dried, raw granulate was coated in a Lödige mixer at ca. 80°C. with melted hydrogenated palm oil and Talc 5/0 M-10; the coating wasapplied in alternating layers as follows (weight percentages relative toraw granulate):

1) 5% w/w of hydrogenated palm oil;

2) 12.5% w/w of talc;

3) 1.0% w/w of hydrogenated palm oil;

4) 5.0% w/w of talc;

5) 2.0% w/w of hydrogenated palm oil;

6) 5.0% w/w of talc.

Granulate 2.

Steps 1-3: As for granulate 1, above, except that the sprayed phytasesolution employed in step 1 contained 65 g of dissolved Neosorb™ 70/70.

Granulate 3.

Steps 1-3: As for granulate 1, above, except that the sprayed phytasesolution employed in step 1 contained 17.5 g of dissolvedpolyvinylpyrrolidone (PVP K30).

Granulate 4.

Steps 1 and 2: As for granulate 2.

Step 3: Omitted

Granulate 4 thus corresponds to granulate 2, but lacks a coating.

EXAMPLE 27

In this example the retention of phytase activity of coatedphytase/cassava starch granulates prepared as in Example 26 was examinedand compared with that for a commercially available phytase-containinggranulate (coated granulate; Phytase Novo CT, obtained from Novo NordiskA/S, Denmark).

Granulates 1-4 as prepared in Example 26, and a Phytase Novo CTgranulate were compared in a so-called pelleting test, according to astandard procedure, at Bioteknologisk Institut, Kolding, Denmark. Inthis test, the respective granulates are mixed with a commercial pigletfeed composition, and the mixture is formulated into pellets underconditions of heat and high humidity. At 950° C., the measured retentionof phytase activity deriving from the three coated, cassava-basedgranulates (granulates 1-3) was very similar (79-84% retention ofactivity), whilst the retention of activity deriving from the commercial(coated) granulate was 61%. The retention of activity deriving from theuncoated cassava-based granulate under the same conditions was approx.52%.

This example thus demonstrates that the formulation of enzymes asgranules based on starch-based cores can result in very substantialprotection of the enzyme content thereof against deactivation underharsh conditions, and that granules of the type in question are verywell suited to the manufacture of feed compositions which require a heattreatment (e.g to ensure removal of pathogenic organisms).

EXAMPLE 28

The granulates (four cassava-based granulates and the Phytase Novo CTgranulate) employed in Example 27 were also tested with respect to easeof “dissolution” (with attendant release of phytase activity) withstirring in an acetate buffer at 37.5° C., starting with phytaseactivities (as granulate) of 50 FYT/nil of buffer. After 60 minutes, allfour cassava-based granulates exhibited more than 90% release of phytaseactivity, whilst the commercial granulate exhibited slightly more than60% release of phytase activity.

This example thus demonstrates, e.g., that starch-based granules of thetype in question possess very advantageous properties with respect tothe availability of the enzyme content thereof under conditions similarto those obtaining in the digestive system of an animal.

What is claimed is:
 1. A process for producing enzyme-containing starch granules, comprising (a) contacting a starch or modified starch core selected from the group consisting of potato starch, sweet-potato starch, and mixtures thereof, with an aqueous enzyme solution or dispersion for sufficient time to result in absorption of the enzyme solution or dispersion into the starch core without agglomeration or disintegration of the starch core; followed by (b) drying the enzyme-containing starch granule.
 2. The process of claim 1, wherein the starch core is intrinsically capable of absorbing at least 5% w/w of water.
 3. The process of claim 1, wherein the starch core is intrinsically capable of absorbing at least 20% w/w of water.
 4. The process of claim 1, wherein the starch core is intrinsically capable of absorbing at least 30% w/w of water.
 5. The process of claim 1, wherein the granule has a ratio between the largest and smallest diameter of less than
 3. 6. The process of claim 1, wherein the ratio between the largest and the smallest diameter of the granule is less than 1.5.
 7. The process of claim 1, wherein the ratio between te largest and the smallest diameter of the granule is equal to or less than 1.2.
 8. The process of claim 1, wherein the starch is gelatinized in a degree range of 2-95%.
 9. The process of claim 1, wherein the degree of starch gelatinization is at least 2%.
 10. The process of claim 8, wherein the degree of starch gelatinization is in the range of 10-60%.
 11. The process of claim 10, wherein the degree of starch gelatinization is in the range of 30-60%.
 12. The process of claim 1, wherein the degree of starch gelatinization is up to 95%.
 13. The process of claim 1, further comprising adding one or more coating layers to the dried enzyme-containing starch granule.
 14. The process of claim 13, comprising adding two or more coating layers.
 15. The process of claim 1, wherein the enzyme-containing starch granule has a size in the range of about 50-4000 μm.
 16. The process of claim 1, wherein the size is in the range of 300-2000 μm.
 17. The process of claim 1, wherein the starch core further comprises one or more materials selected from the group consisting of binders, fillers, plasticizers, fibrous materials and superabsorbents.
 18. The process of claim 1, wherein the aqueous enzyme solution or dispersion comprises one or more enzymes selected from the group consisting of peptidases, amylases, lipases, cellulases, oxidoreductases, phytases, and xylanases.
 19. An enzyme-containing starch granule produced by the process of claim
 1. 20. An animal feed composition comprising the enzyme-containing granule of claim
 19. 